U.S. patent application number 16/362940 was filed with the patent office on 2019-11-14 for predicting response to pd-1 axis inhibitors.
This patent application is currently assigned to Hoffmann-La Roche Inc.. The applicant listed for this patent is Hoffmann-La Roche Inc.. Invention is credited to Christian KLEIN, Maud Lea MAYOUX, Andreas ROLLER, Wei XU.
Application Number | 20190346444 16/362940 |
Document ID | / |
Family ID | 60019875 |
Filed Date | 2019-11-14 |
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United States Patent
Application |
20190346444 |
Kind Code |
A1 |
KLEIN; Christian ; et
al. |
November 14, 2019 |
PREDICTING RESPONSE TO PD-1 AXIS INHIBITORS
Abstract
The invention is concerned with a method of predicting response
to a PD-1 axis inhibitor such as anti-PD-L1 antibody by determining
the abundance of dendritic cells (DCs) in a tumor tissue sample.
The abundance of DCs characterized by enhanced expressions of XCR1,
IRF8, BATF3 and FLT3 predicts clinical response to the PD-L1
blockade treatment.
Inventors: |
KLEIN; Christian;
(Schlieren, CH) ; MAYOUX; Maud Lea; (Schlieren,
CH) ; ROLLER; Andreas; (Basel, CH) ; XU;
Wei; (Schlieren, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hoffmann-La Roche Inc. |
Little Falls |
NJ |
US |
|
|
Assignee: |
Hoffmann-La Roche Inc.
Little Falls
NJ
|
Family ID: |
60019875 |
Appl. No.: |
16/362940 |
Filed: |
March 25, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/EP2017/074150 |
Sep 25, 2017 |
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16362940 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/563 20130101;
A61P 43/00 20180101; G01N 33/57423 20130101; A61P 35/00 20180101;
G01N 2800/52 20130101; G01N 33/57407 20130101 |
International
Class: |
G01N 33/574 20060101
G01N033/574; G01N 33/563 20060101 G01N033/563 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 26, 2016 |
EP |
16190591.4 |
Apr 18, 2017 |
EP |
17166789.2 |
Claims
1. A method for treating a patient with cancer, the method
comprising: (i) determining in vitro the abundance of dendritic
cells (DCs) in a tumor tissue sample obtained from the patient with
cancer; (ii) identifying the patient as being responsive to a
therapy comprising an effective amount of a PD-1 axis inhibitor
based on (i); and (iii) treating or recommending treatment of the
patient with the therapy comprising an effective amount of a PD-1
axis inhibitor based on (ii).
2. The method of claim 1, wherein the abundance of DCs is
characterized by an expression level of one or more genes selected
from the group consisting of XCR1, IRF8, BATF3 and FLT3.
3. The method of claim 2, wherein the method further comprises a
step of comparing the expression level of the one or more genes to
a reference level, whereby an increased expression level is
indicative of response to a therapy comprising a PD-1 axis
inhibitor.
4. The method of claim 2, wherein the expression level is detected
in the sample by protein expression.
5. The method of claim 2, wherein the expression level is detected
in the sample by mRNA expression.
6. The method of claim 2, wherein the expression level is detected
using a method selected from the group consisting of FACS, Western
blot, ELISA, immunoprecipitation, immunohistochemistry,
immunofluorescence, radioimmunoassay, immunodetection methods, mass
spectrometery, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR,
RNA-seq, microarray analysis, nanostring, SAGE, MassARRAY
technique, and FISH, and combinations thereof.
7. The method of claim 1, wherein the cancer is selected from the
group consisting of non-small cell lung cancer, small cell lung
cancer, renal cell cancer, colorectal cancer, ovarian cancer,
breast cancer, pancreatic cancer, gastric carcinoma, bladder
cancer, esophageal cancer, mesothelioma, melanoma, head and neck
cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma,
cervical cancer, thymic carcinoma, leukemia, lymphomas, myelomas,
mycoses fungoids, merkel cell cancer, and other hematologic
malignancies.
8. The method of claim 1, wherein the therapy includes a PD-1 axis
inhibitor as monotherapy.
9. The method of claim 1, wherein the therapy further comprises an
effective amount of a second agent selected from the group
consisting of cytotoxic agent, a chemotherapeutic agent, a growth
inhibitory agent, a radiation therapy agent, and anti-angiogenic
agent, and combinations thereof.
10. The method of claim 1, wherein the PD-1 axis inhibitor is a
PD-1 binding antagonist.
11. The method of claim 10, wherein the PD-1 binding antagonist
inhibits the binding of PD-1 to PD-L1.
12. The method of claim 10, wherein the PD-1 binding antagonist is
an anti-PD-1 antibody.
13. The method of claim 1, wherein the PD-1 axis inhibitor is a
PD-L1 binding antagonist.
14. The method of claim 13, wherein the PD-L1 binding antagonist
inhibits the binding of PD-L1 to PD-1.
15. The method of claim 13, wherein PD-L1 binding antagonist is an
anti-PD-L1 antibody.
16. The method of claim 15, wherein the anti-PD-L1 antibody is an
antibody fragment selected from the group consisting of Fab,
Fab'-SH, Fv, scFv, and (Fab')2 fragments.
17. The method of claim 15, wherein the anti-PD-L1 antibody is
selected from the group consisting of YW243.55.S70, MPDL3280A,
MDX-1105, and MEDI4736.
18. The method of claim 1, wherein the tumor tissue sample is a
sample obtained from the patient prior to the therapy with a PD-1
axis inhibitor.
19. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of International
Application No. PCT/EP2017/074150 having an International Filing
Date of 25 Sep. 2017, claiming priority to application numbers EP
16190591.4 filed 26 Sep. 2016 and EP 17166789.2 filed 18 Apr. 2017,
each of which are incorporated herein by reference in its
entirety.
SEQUENCE LISTING
[0002] This application contains a Sequence Listing which has been
submitted electronically in ASCII format and is hereby incorporated
by reference in its entirety. Said ASCII copy, created on Mar. 5,
2019, is named P33856-US_Sequence_Listing.txt and is 9,430 bytes in
size.
FIELD OF THE INVENTION
[0003] The present invention relates to a biomarker for predicting
response of a patient with a cancer to a PD-1 axis inhibitor such
as an anti-PD-L1 antibody. Provided herein is a method of
identifying a cancer patient responsive to a PD-1 axis inhibitor by
determining the abundance of dendritic cells (DCs) in a tumor
tissue sample.
BACKGROUND OF THE INVENTION
[0004] Myeloid cells including DCs and macrophages are crucial in
initiating adaptive immune response via priming of naive T cells to
educate and generate effector cells. DCs, the professional
antigen-presenting cells (APC), are bone marrow-derived cells that
populate all lymphoid organs as well as nearly all non-lymphoid
organs. Pathogen-sensed mature DCs in the periphery express high
levels of MHC (class I and II) and costimulatory molecules, and are
able to migrate to the secondary lymphoid organs to stimulate naive
T cells to induce adaptive immunity.
[0005] On the other hand, many myeloid cell subsets are able to
infiltrate the tumor microenvironment, and depending on the
activation status, they provide tumor escaping mechanism via
immune-suppression or direct tumoricidal activities. Various
markers can reflect the state of DCs and macrophages activation,
one example of activating receptor is the CD40 protein. Antibodies
agonizing the CD40 receptor have shown anti-tumor effects in both
preclinical and clinical settings by activating the DCs to induce T
cell infiltration into the tumor and by polarizing macrophages to
kill tumors (Beatty et al., 2011, Science, 331: 1612-6). On the
contrary, Myeloid-derived suppressor cells (MDSC) have been
identified to play a negative role in the tumor microenvironment by
promoting immune-suppressive responses in several solid cancers as
shown in human melanoma (Gorgun et al., 2013, Blood, 121: 2975-87).
Therefore a more comprehensive understanding of how the
immunosuppressive milieu develops and persists is critical to guide
the success of full power new immunotherapies.
[0006] PD-1 is an immunoglobulin superfamily member discovered on
1992 as a gene up-regulated in T cell hybridoma undergoing cell
death (Ishida et al., 1992, EMBO J, 11: 3887-95). PD-1 is mainly
found on activated T, B and myeloid cells. The important negative
regulatory function of PD-1 was revealed by autoimmune-prone
phenotype of Pdcd1-/- mice in 1999 (Nishimura et al., 1999,
Immunity, 11: 141-51). In 1999 PD-L1 (B7-H1), the first ligand of
PD-1, was identified (Dong et al., 1999, Nat Med, 5: 1365-9),
followed by PD-L2 (B7-DC) in 2001 (Latchman et al., 2001, Nat
Immunol, 2: 261-8). Another costimulatory molecule, the CD80 (B7-1)
interacts specifically with PD-L1 (Butte et al., 2007, Immunity,
27: 111-22) as well. PD-1 contains two immunoreceptor
tyrosine-based motifs that are phosphorylated upon receptor
engagement and recruit Src homology 2-domain-containing tyrosine
phosphatase 2. The PD-1:PD-L1 pathway inhibits T cell proliferation
by reducing the production of IL-2 and restricts the number of T
cells that gain entry into the cell cycle as well as their
subsequent division rate. Up-regulation of PD-L1 expression was
described in several human tumors types, which hijacks the PD-L1 to
interact with PD-1 on T cells and suppress effector function. These
findings led to the successful clinical application of PD-1
blockade in treating solid tumors (Sharma et al., 2015, Cell, 161:
205-14). Nevertheless, so far only a minor subset of patients
(<30%) benefit from such a therapy, with as-yet unknown
mechanisms (Zou et al., 2016, Sci Transl Med, 8: 328rv4).
[0007] Accordingly, there is a need for methods for determining
which patients respond particularly well to a therapy with a PD-1
axis inhibitor such as an anti-PD-L1 antibody that inhibits the
binding of PD-L1 to PD-1.
SUMMARY OF THE INVENTION
[0008] Despite intensive research on the role of PD-1 in
lymphocytes, little has been studied to unravel the molecular
regulation of PD-1/PD-L1 pathway on myeloid cells, particularly on
DCs, and the significance of this pathway blockade in regulating
tumor immunity. Depending on the activating signals, certain
subpopulations of DCs are able to suppress immune responses by
establishing and maintaining T cell tolerance (Dhodapkar et al.,
2001, J Exp Med, 193: 233-8; Steinman et al., 2003, Annu Rev
Immunol, 21: 685-711; Jonuleit et al., 2001., Trends Immunol, 22:
394-400). Immunosuppressive DCs were found in the tumor
microenvironment (Scarlett et al., 2012., J Exp Med, 209: 495-506).
Interestingly, tumor-infiltrating DCs became PD-1 positive over the
course of ovarian cancer progression (Krempski et al., 2011., J
Immunol, 186: 6905-13), and it seems likely that PD-1 blocked
Nuclear Factor-kappa B-dependent activation to render the DCs
immunosuppressive (Karyampudi et al., 2016, Cancer Res, 76:
239-50). Earlier studies have indicated that blocking PD-L1 on
human DCs in vitro enhanced T cell immunity (Brown et al., 2003, J
Immunol, 170: 1257-66). It was suggested that PD-1 negatively
regulates murine DCs in vivo (Krempski et al. 2011; Park et al.,
2014, J Leukoc Biol, 95: 621-9; Yao et al., 2009, Blood, 113:
5811-8). Given that PD-L1 binds to both CD80 and PD-1 (Keir et al.,
2008, Annu Rev Immunol, 26: 677-704), and DCs express all these
three receptors/ligands simultaneously, the inventors hypothesized
that PD-1/PD-L1 is regulated in a controlled manner, thus
immunotherapies targeting this pathway could hold a yet
underappreciated mechanism of actions by modulating DCs function
and/or other myeloid cell subset that influence the downstream T
cell lineage development.
[0009] Provided herein is evidence that DCs are the primary targets
of PD-L1 blockade enabling enhanced anti-tumor immunity. It is
shown that human DCs express both PD-1 and PD-L1, and the PD-1 is
negatively regulated upon activation by DCs. Further, PD-L1
blockade directly activate DCs, rendering them acquire enhanced
capacity to activate T cells, both in human in vitro settings and
in tumor-bearing mice. Depleting DCs in mice where tumor has been
established showed a compromised response to PD-L1 blockade
treatment, suggesting a direct contribution of DCs to PD-L1
blockade-mediated anti-tumor immunity. Moreover, the analysis of
tumor biopsy at baseline from patients with renal cell carcinoma
who received treatment with an anti-PD-L1 antibody, atezolizumab,
and showed that patients with higher expressions of genes related
to development and function of DCs had a significant survival
advantage as compared to those with lower expressions. Thus, the
data support that PD-L1 blockade directly targets DCs to enhance
anti-tumor immunity. The abundance of functional DCs in tumor
tissue is predictor of a better clinical outcome in response to a
therapy with a PD-1 axis inhibitor such as PD-L1 blockade
treatment.
[0010] It is further demonstrated herein that, upon maturation of
DCs, PD-1 expression is down-regulated. However, PD-L1 expression
increases, which leads to binding of PD-L1 to CD80 on the surface
of DCs, sequestering CD80 and preventing binding of CD80 to CD28
for co-stimulation on T cells. Administration of PD-L1 antibodies
relieves the CD80 sequestration, enabling further co-stimulation of
anti-cancer T cells through CD80/CD28 interaction. This represents
the first demonstration of how the PD-L1/PD-1 pathway biologically
inhibits DCs in tumor, and functions as an immune checkpoint in
anti-cancer T cell priming and activation.
[0011] Provided herein therefore are a method of predicting
clinical response to a PD-1 axis inhibitor in a patient with cancer
and a pharmaceutical composition comprising a PD1 axis inhibitor
for use in treatment of a patient with cancer who is likely to
respond to a PD-1 axis inhibitor.
[0012] The following numbered paragraphs (para.) define some
embodiments of the present invention.
[0013] 1. An in vitro method of identifying a patient with cancer
who is responsive to a therapy comprising an effective amount of a
PD-1 axis inhibitor, the method comprising determining the
abundance of dendritic cells (DCs) in a tumor tissue sample
obtained from a patient with cancer.
[0014] 2. The method of para 1, wherein the abundance of DCs is
characterized by an expression level of one or more genes selected
from the group consisting of XCR1, IRF8, BATF3 and FLT3.
[0015] 3. The method of para. 2, wherein the method further
comprises a step of comparing the expression level of the one or
more genes to a reference level, whereby an increased expression
level is indicative of response to a therapy comprising a PD-1 axis
inhibitor.
[0016] 4. The method of para. 2 or 3, wherein the expression level
is detected in the sample by protein expression.
[0017] 5. The method of para. 2 or 3, wherein the expression level
is detected in the sample by mRNA expression.
[0018] 6. The method of any one of para. 2 to 5, wherein the
expression level is detected using a method selected from the group
consisting of FACS, Western blot, ELISA, immunoprecipitation,
immunohistochemistry, immunofluorescence, radioimmunoas say,
immunodetection methods, mass spectrometery, HPLC, qPCR, RT-qPCR,
multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis,
nanostring, SAGE, MassARRAY technique, and FISH, and combinations
thereof.
[0019] 7. The method of any one of para. 1 to 6, wherein the cancer
is selected from the group consisting of non-small cell lung
cancer, small cell lung cancer, renal cell cancer, colorectal
cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric
carcinoma, bladder cancer, esophageal cancer, mesothelioma,
melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate
cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia,
lymphomas, myelomas, mycoses fungoids, merkel cell cancer, and
other hematologic malignancies.
[0020] 8. The method of any one of para. 1 to 7, wherein the
therapy includes a PD-1 axis inhibitor as monotherapy.
[0021] 9. The method of any one of para. 1 to 7, wherein the
therapy further comprises an effective amount of a second agent
selected from the group consisting of cytotoxic agent, a
chemotherapeutic agent, a growth inhibitory agent, a radiation
therapy agent, and anti-angiogenic agent, and combinations
thereof.
[0022] 10. The method of any one of para. 1 to 9, wherein the PD-1
axis inhibitor is a PD-1 binding antagonist.
[0023] 11. The method of para. 10, wherein the PD-1 binding
antagonist inhibits the binding of PD-1 to PD-L1.
[0024] 12. The method of para. 10 or 11, wherein the PD-1 binding
antagonist is an anti-PD-1 antibody.
[0025] 13. The method of any one of para. 1 to 9, wherein the PD-1
axis inhibitor is a PD-L1 binding antagonist.
[0026] 14. The method of para. 13, wherein the PD-L1 binding
antagonist inhibits the binding of PD-L1 to PD-1.
[0027] 15. The method of para. 13 or 14, wherein PD-L1 binding
antagonist is an anti-PD-L1 antibody.
[0028] 16. The method of para. 15, wherein the anti-PD-L1 antibody
is an antibody fragment selected from the group consisting of Fab,
Fab'-SH, Fv, scFv, and (Fab')2 fragments.
[0029] 17. The method of para. 15 or 16, wherein the anti-PD-L1
antibody is selected from the group consisting of YW243.55.570,
MPDL3280A, MDX-1105, and MEDI4736.
[0030] 18. The method of any one of para. 1 to 17, wherein the
tumor tissue sample is a sample obtained from the patient prior to
the therapy with a PD-1 axis inhibitor.
[0031] 19. A pharmaceutical composition comprising a PD-1 axis
inhibitor for use in the treatment of a patient with cancer,
wherein the patient is determined to be responsive to a therapy
comprising an effective amount of a PD-1 axis inhibitor in
accordance with the method of any one of para. 1 to 18.
[0032] In some embodiments, the present invention relates to a
method of determining whether a patient with cancer is more
suitably treated by a therapy comprising an effective amount of a
PD-1 axis inhibitor, the method comprising determining the
abundance of DCs in a tumor tissue sample obtained from a patient
with cancer.
[0033] In some embodiments, the present invention relates to a
method of improving the treatment effect of a therapy comprising an
effective amount of a PD-1 axis inhibitor in a patient with cancer,
the method comprising determining the abundance of DCs in a tumor
tissue sample obtained from a patient with cancer.
[0034] In some embodiments, the present invention relates to a
method of treating a patient with cancer. The method comprises
administering to a patient with cancer a therapy comprising an
effective amount of a PD-1 axis inhibitor, the method comprising
determining the abundance of DCs in a tumor tissue sample obtained
from the patient.
[0035] These and other embodiments are further described in the
detailed description below.
BRIEF DESCRIPTION OF THE FIGURES
[0036] FIGS. 1A-1B show the immunostaining of PD-1 and PD-L1
expression on human DCs. In vitro-generated DCs express both PD-1
and PD-L1 (FIG. 1A), and the expression profile of PD-1 and PD-L1
is negatively correlated (FIG. 1B).
[0037] FIGS. 2A-2B show that upon maturation of DCs, PD-1 is
downregulated while PD-L1 is upregulated (FIGS. 2A and 2B).
[0038] FIGS. 3A-3B show the results of the immunostaining of
immature DCs (iDCs), mature DCs (mDCs) and tolerogenic DCs (tDCs)
(FIG. 3A) and the flow cytometry measurement of of T cell
proliferation in T cells co-cultured with different DCs (FIG. 3B).
These figures show that the expression profile of PD-1 is
negatively correlated with T cell stimulatory capacity.
[0039] FIGS. 4A-4B show that DCs pre-incubated with an anti-PD-L1
Ab acquired enhanced T cell stimulatory capacity (FIG. 4A),
accompanied with increased IFN-.gamma. production (FIG. 4B).
[0040] FIGS. 5A-5C shows tumor-bearing mice received PD-L1 Ab
showed increased frequency of DCs in spleen and draining-lymph
nodes (DLN) as compared to the vehicle group (FIGS. 5A-B), and the
DCs (gated on CD11c+ cells) showed higher expression of CD86, a
marker of activation/maturation (FIG. 5C).
[0041] FIG. 6A shows the experimental design of an in vivo study of
using CD11c-DTR mice where CD11c.sup.+ DCs can be depleted by
administration of diphtheria toxin (DT) prior to the treatment of
an anti-PD-L1 antibody. FIG. 6B shows that anti-PD-L1
antibody-mediated tumor growth inhibition is compromised in mice
that DCs were depleted by DT.
[0042] FIG. 7 summarizes clinical response to atezolizumab in
patients with renal cell carcinoma.
[0043] FIGS. 8A-8B show the Kaplan-Meier survival curves in
patients with RCC. Expression of genes related to DC development
correlates with the survival advantages by a PD-1 axis inhibitor
atezolizumab.
[0044] FIG. 9 summarizes a list of genes related to DCs and the
correlative response to the Kaplan-Meier survival curve.
[0045] FIG. 10 shows the Kaplan-Meier survival curves based on the
expression of a cumulative DC gene signature including genes: IRF8,
FLT3, BATF3 and XCR1. A higher DC signature score correlates with
clinical benefit to a PD-1 axis inhibitor atezolizumab in patients
with RCC.
[0046] FIG. 11 shows that DC-related gene signature correlates with
survival in patients with NSCLC treated with atezolizumab.
[0047] FIG. 12 shows that DC-related gene signature correlates with
survival in PD-L1.sup.+ patients with NSCLC treated with
atezolizumab.
DETAILED DESCRIPTION OF THE EMBODIMENTS
Definitions
[0048] The term "PD-1 axis inhibitor" is a molecule that inhibits
the interaction of a PD-1 axis binding partner with either one or
more of its binding partner, so as to remove T-cell dysfunction
resulting from signaling on the PD-1 signaling axis--with a result
being to restore or enhance T-cell function, e.g., proliferation,
cytokine production, target cell killing. As used herein, a PD-1
axis inhibitor includes a PD-1 binding antagonist and a PD-L1
binding antagonist.
[0049] The term "PD-1 binding antagonist" is a molecule that
decreases, blocks, inhibits, abrogates or interferes with signal
transduction resulting from the interaction of PD-1 with one or
more of its binding partners, such as PD-L1, PD-L2. In some
embodiments, the PD-1 binding antagonist is a molecule that
inhibits the binding of PD-1 to its binding partners. In a specific
aspect, the PD-1 binding antagonist inhibits the binding of PD-1 to
PD-L1 and/or PD-L2. For example, PD-1 binding antagonists include
anti-PD-1 antibodies, antigen binding fragments thereof,
immunoadhesins, fusion proteins, oligopeptides and other molecules
that decrease, block, inhibit, abrogate or interfere with signal
transduction resulting from the interaction of PD-1 with PD-L1
and/or PD-L2. In one embodiment, a PD-1 binding antagonist reduces
the negative co-stimulatory signal mediated by or through cell
surface proteins expressed on T lymphocytes mediated signaling
through PD-1 so as render a dysfunctional T-cell less
dysfunctional, e.g. enhancing effector responses to antigen
recognition. In some embodiments, the PD-1 binding antagonist is an
anti-PD-1 antibody. In a specific aspect, a PD-1 binding antagonist
is MDX-1106 described herein. In another specific aspect, a PD-1
binding antagonist is Merck 3745 described herein.
[0050] The term "PD-L1 binding antagonist" is a molecule that
decreases, blocks, inhibits, abrogates or interferes with signal
transduction resulting from the interaction of PD-L1 with either
one or more of its binding partners, such as PD-1, B7-1. In some
embodiments, a PD-L1 binding antagonist is a molecule that inhibits
the binding of PD-L1 to its binding partners. In a specific aspect,
the PD-L1 binding antagonist inhibits binding of PD-L1 to PD-1
and/or B7-1. In some embodiments, the PD-L1 binding antagonists
include anti-PD-L1 antibodies, antigen binding fragments thereof,
immunoadhesins, fusion proteins, oligopeptides and other molecules
that decrease, block, inhibit, abrogate or interfere with signal
transduction resulting from the interaction of PD-L1 with one or
more of its binding partners, such as PD-1 and B7-1. In one
embodiment, a PD-L1 binding antagonist reduces the negative
co-stimulatory signal mediated by or through cell surface proteins
expressed on T lymphocytes mediated signaling through PD-L1 so as
to render a dysfunctional T-cell less dysfunctional, e.g. enhancing
effector responses to antigen recognition. In some embodiments, a
PD-L1 binding antagonist is an anti-PD-L1 antibody. In a specific
aspect, an anti-PD-L1 antibody is YW243.55.570 described herein. In
another specific aspect, an anti-PD-L1 antibody is MDX-1105
described herein. In still another specific aspect, an anti-PD-L1
antibody is MPDL3280A described herein.
[0051] The term "antibody" herein is used in the broadest sense and
encompasses various antibody structures, including but not limited
to monoclonal antibodies, polyclonal antibodies, multispecific
antibodies, e.g., bispecific antibodies, and antibody fragments so
long as they exhibit the desired antigen-binding activity.
[0052] The terms "anti-PD-L1 antibody" and "an antibody that binds
to PD-L1" refer to an antibody that is capable of binding PD-L1
with sufficient affinity such that the antibody is useful as a
diagnostic and/or therapeutic agent in targeting PD-L1. In one
embodiment, the extent of binding of an anti-PD-L1 antibody to an
unrelated, non-PD-L1 protein is less than about 10% of the binding
of the antibody to PD-L1 as measured, e.g., by a radioimmunoassay
(RIA). In certain embodiments, an anti-PD-L1 antibody binds to an
epitope of PD-L1 that is conserved among PD-L1 from different
species.
[0053] A "blocking" antibody or an "antagonist" antibody is one
which inhibits or reduces biological activity of the antigen it
binds. Preferred blocking antibodies or antagonist antibodies
substantially or completely inhibit the biological activity of the
antigen.
[0054] An "antibody fragment" refers to a molecule other than an
intact antibody that comprises a portion of an intact antibody that
binds the antigen to which the intact antibody binds. Examples of
antibody fragments include but are not limited to Fv, Fab, Fab',
Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain
antibody molecules (e.g., scFv); and multispecific antibodies
formed from antibody fragments.
[0055] An "antibody that binds to the same epitope" as a reference
antibody refers to an antibody that blocks binding of the reference
antibody to its antigen in a competition assay by 50% or more, and
conversely, the reference antibody blocks binding of the antibody
to its antigen in a competition assay by 50% or more. An exemplary
competition assay is provided herein.
[0056] The term "chimeric" antibody refers to an antibody in which
a portion of the heavy and/or light chain is derived from a
particular source or species, while the remainder of the heavy
and/or light chain is derived from a different source or
species.
[0057] The terms "full length antibody," "intact antibody," and
"whole antibody" are used herein interchangeably to refer to an
antibody having a structure substantially similar to a native
antibody structure or having heavy chains that contain an Fc region
as defined herein.
[0058] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical and/or bind the same epitope, except for
possible variant antibodies, e.g., containing naturally occurring
mutations or arising during production of a monoclonal antibody
preparation, such variants generally being present in minor
amounts. In contrast to polyclonal antibody preparations, which
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody of a monoclonal
antibody preparation is directed against a single determinant on an
antigen. Thus, the modifier "monoclonal" indicates the character of
the antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by a variety of techniques, including but not
limited to the hybridoma method, recombinant DNA methods,
phage-display methods, and methods utilizing transgenic animals
containing all or part of the human immunoglobulin loci, such
methods and other exemplary methods for making monoclonal
antibodies being described herein.
[0059] A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human or a human cell or derived from a non-human source that
utilizes human antibody repertoires or other human
antibody-encoding sequences. This definition of a human antibody
specifically excludes a humanized antibody comprising non-human
antigen-binding residues.
[0060] A "humanized" antibody refers to a chimeric antibody
comprising amino acid residues from non-human HVRs and amino acid
residues from human FRs. In certain embodiments, a humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the HVRs (e.g., CDRs) correspond to those of a non-human
antibody, and all or substantially all of the FRs correspond to
those of a human antibody. A humanized antibody optionally may
comprise at least a portion of an antibody constant region derived
from a human antibody. A "humanized form" of an antibody, e.g., a
non-human antibody, refers to an antibody that has undergone
humanization.
[0061] The term "detection" includes any means of detecting,
including direct and indirect detection.
[0062] The term "biomarker" as used herein refers to an indicator,
e.g., predictive, diagnostic, and/or prognostic, which can be
detected in a sample. The biomarker may serve as an indicator of a
particular subtype of a disease or disorder (e.g., cancer)
characterized by certain, molecular, pathological, histological,
and/or clinical features. In some embodiments, a biomarker is a
gene. Biomarkers include, but are not limited to, polynucleotides
(e.g., DNA, and/or RNA), polynucleotide copy number alterations
(e.g., DNA copy numbers), polypeptides, polypeptide and
polynucleotide modifications (e.g. posttranslational
modifications), carbohydrates, and/or glycolipid-based molecular
markers.
[0063] The terms "biomarker signature," "signature," "biomarker
expression signature," or "expression signature" are used
interchangeably herein and refer to one or a combination of
biomarkers whose expression is an indicator, e.g., predictive,
diagnostic, and/or prognostic. The biomarker signature may serve as
an indicator of a particular subtype of a disease or disorder
(e.g., cancer) characterized by certain molecular, pathological,
histological, and/or clinical features. In some embodiments, the
biomarker signature is a "gene signature." The term "gene
signature" is used interchangeably with "gene expression signature"
and refers to one or a combination of polynucleotides whose
expression is an indicator, e.g., predictive, diagnostic, and/or
prognostic. In some embodiments, the biomarker signature is a
"protein signature." The term "protein signature" is used
interchangeably with "protein expression signature" and refers to
one or a combination of polypeptides whose expression is an
indicator, e.g., predictive, diagnostic, and/or prognostic.
[0064] The "amount" or "level" of a biomarker associated with an
increased clinical benefit to an individual is a detectable level
in a biological sample. These can be measured by methods known to
one skilled in the art and also disclosed herein. The expression
level or amount of biomarker assessed can be used to determine the
response to the treatment.
[0065] The terms "level of expression" or "expression level" in
general are used interchangeably and generally refer to the amount
of a biomarker in a biological sample. "Expression" generally
refers to the process by which information (e.g., gene-encoded
and/or epigenetic) is converted into the structures present and
operating in the cell. Therefore, as used herein, "expression" may
refer to transcription into a polynucleotide, translation into a
polypeptide, or even polynucleotide and/or polypeptide
modifications (e.g., posttranslational modification of a
polypeptide). Fragments of the transcribed polynucleotide, the
translated polypeptide, or polynucleotide and/or polypeptide
modifications (e.g., posttranslational modification of a
polypeptide) shall also be regarded as expressed whether they
originate from a transcript generated by alternative splicing or a
degraded transcript, or from a post-translational processing of the
polypeptide, e.g., by proteolysis. "Expressed genes" include those
that are transcribed into a polynucleotide as mRNA and then
translated into a polypeptide, and also those that are transcribed
into RNA but not translated into a polypeptide (for example,
transfer and ribosomal RNAs).
[0066] The term "reference level" herein refers to a predetermined
value. As a skilled person will appreciate the reference level is
predetermined and set to meet the requirements in terms of e.g.
specificity and/or sensitivity. These requirements can vary, e.g.
from regulatory body to regulatory body. It may for example be that
assay sensitivity or specificity, respectively, has to be set to
certain limits, e.g. 80%, 90% or 95%. These requirements may also
be defined in terms of positive or negative predictive values.
Nonetheless, based on the teaching given in the present invention
it will always be possible to arrive at the reference level meeting
those requirements. In one embodiment the reference level is
determined in healthy individuals. The reference value in one
embodiment has been predetermined in the disease entity to which
the patient belongs. In certain embodiments the reference level can
e.g. be set to any percentage between 25% and 75% of the overall
distribution of the values in a disease entity investigated. In
other embodiments the reference level can e.g. be set to the
median, tertiles or quartiles as determined from the overall
distribution of the values in a disease entity investigated. In one
embodiment the reference level is set to the median value as
determined from the overall distribution of the values in a disease
entity investigated.
[0067] In certain embodiments, the term "increase", "increased" or
"above" refers to a level above the reference level.
[0068] "Amplification," as used herein generally refers to the
process of producing multiple copies of a desired sequence.
"Multiple copies" mean at least two copies. A "copy" does not
necessarily mean perfect sequence complementarity or identity to
the template sequence. For example, copies can include nucleotide
analogs such as deoxyinosine, intentional sequence alterations
(such as sequence alterations introduced through a primer
comprising a sequence that is hybridizable, but not complementary,
to the template), and/or sequence errors that occur during
amplification.
[0069] The term "multiplex-PCR" refers to a single PCR reaction
carried out on nucleic acid obtained from a single source (e.g., an
individual) using more than one primer set for the purpose of
amplifying two or more DNA sequences in a single reaction.
[0070] "Stringency" of hybridization reactions is readily
determinable by one of ordinary skill in the art, and generally is
an empirical calculation dependent upon probe length, washing
temperature, and salt concentration. In general, longer probes
require higher temperatures for proper annealing, while shorter
probes need lower temperatures. Hybridization generally depends on
the ability of denatured DNA to reanneal when complementary strands
are present in an environment below their melting temperature. The
higher the degree of desired homology between the probe and
hybridizable sequence, the higher the relative temperature which
can be used. As a result, it follows that higher relative
temperatures would tend to make the reaction conditions more
stringent, while lower temperatures less so. For additional details
and explanation of stringency of hybridization reactions, see
Ausubel et al., Current Protocols in Molecular Biology, Wiley
Interscience Publishers, (1995).
[0071] "Stringent conditions" or "high stringency conditions", as
defined herein, can be identified by those that: (1) employ low
ionic strength and high temperature for washing, for example 0.015
M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl
sulfate at 50.degree. C.; (2) employ during hybridization a
denaturing agent, such as formamide, for example, 50% (v/v)
formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%
polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with
750 mM sodium chloride, 75 mM sodium citrate at 42.degree. C.; or
(3) overnight hybridization in a solution that employs 50%
formamide, 5.times.SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM
sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate,
5.times.Denhardt's solution, sonicated salmon sperm DNA (50
.mu.g/ml), 0.1% SDS, and 10% dextran sulfate at 42.degree. C., with
a 10 minute wash at 42.degree. C. in 0.2.times.SSC (sodium
chloride/sodium citrate) followed by a 10 minute high-stringency
wash consisting of 0.1.times.SSC containing EDTA at 55.degree.
C.
[0072] "Moderately stringent conditions" can be identified as
described by Sambrook et al., Molecular Cloning: A Laboratory
Manual, New York: Cold Spring Harbor Press, 1989, and include the
use of washing solution and hybridization conditions (e.g.,
temperature, ionic strength and % SDS) less stringent that those
described above. An example of moderately stringent conditions is
overnight incubation at 37.degree. C. in a solution comprising: 20%
formamide, 5.times.SSC (150 mM NaCl, 15 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times.Denhardt's solution, 10%
dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA,
followed by washing the filters in 1.times.SSC at about
37-50.degree. C. The skilled artisan will recognize how to adjust
the temperature, ionic strength, etc. as necessary to accommodate
factors such as probe length and the like.
[0073] The technique of "polymerase chain reaction" or "PCR" as
used herein generally refers to a procedure wherein minute amounts
of a specific piece of nucleic acid, RNA and/or DNA, are amplified
as described in U.S. Pat. No. 4,683,195 issued 28 Jul. 1987.
Generally, sequence information from the ends of the region of
interest or beyond needs to be available, such that oligonucleotide
primers can be designed; these primers will be identical or similar
in sequence to opposite strands of the template to be amplified.
The 5' terminal nucleotides of the two primers may coincide with
the ends of the amplified material. PCR can be used to amplify
specific RNA sequences, specific DNA sequences from total genomic
DNA, and cDNA transcribed from total cellular RNA, bacteriophage or
plasmid sequences, etc. See generally Mullis et al., Cold Spring
Harbor Symp. Quant. Biol., 51: 263 (1987); Erlich, ed., PCR
Technology, (Stockton Press, N.Y., 1989). As used herein, PCR is
considered to be one, but not the only, example of a nucleic acid
polymerase reaction method for amplifying a nucleic acid test
sample, comprising the use of a known nucleic acid (DNA or RNA) as
a primer and utilizes a nucleic acid polymerase to amplify or
generate a specific piece of nucleic acid or to amplify or generate
a specific piece of nucleic acid which is complementary to a
particular nucleic acid.
[0074] "Quantitative real time polymerase chain reaction" or
"qRT-PCR" refers to a form of PCR wherein the amount of PCR product
is measured at each step in a PCR reaction. This technique has been
described in various publications including Cronin et al., Am. J.
Pathol. 164(1):35-42 (2004); and Ma et al., Cancer Cell 5:607-616
(2004).
[0075] The term "microarray" refers to an ordered arrangement of
hybridizable array elements, preferably polynucleotide probes, on a
substrate.
[0076] The term "polynucleotide," when used in singular or plural,
generally refers to any polyribonucleotide or
polydeoxyribonucleotide, which may be unmodified RNA or DNA or
modified RNA or DNA. Thus, for instance, polynucleotides as defined
herein include, without limitation, single- and double-stranded
DNA, DNA including single- and double-stranded regions, single- and
double-stranded RNA, and RNA including single- and double-stranded
regions, hybrid molecules comprising DNA and RNA that may be
single-stranded or, more typically, double-stranded or include
single- and double-stranded regions. In addition, the term
"polynucleotide" as used herein refers to triple-stranded regions
comprising RNA or DNA or both RNA and DNA. The strands in such
regions may be from the same molecule or from different molecules.
The regions may include all of one or more of the molecules, but
more typically involve only a region of some of the molecules. One
of the molecules of a triple-helical region often is an
oligonucleotide. The term "polynucleotide" specifically includes
cDNAs. The term includes DNAs (including cDNAs) and RNAs that
contain one or more modified bases. Thus, DNAs or RNAs with
backbones modified for stability or for other reasons are
"polynucleotides" as that term is intended herein. Moreover, DNAs
or RNAs comprising unusual bases, such as inosine, or modified
bases, such as tritiated bases, are included within the term
"polynucleotides" as defined herein. In general, the term
"polynucleotide" embraces all chemically, enzymatically and/or
metabolically modified forms of unmodified polynucleotides, as well
as the chemical forms of DNA and RNA characteristic of viruses and
cells, including simple and complex cells.
[0077] The term "oligonucleotide" refers to a relatively short
polynucleotide, including, without limitation, single-stranded
deoxyribonucleotides, single- or double-stranded ribonucleotides,
RNA:DNA hybrids and double-stranded DNAs. Oligonucleotides, such as
single-stranded DNA probe oligonucleotides, are often synthesized
by chemical methods, for example using automated oligonucleotide
synthesizers that are commercially available. However,
oligonucleotides can be made by a variety of other methods,
including in vitro recombinant DNA-mediated techniques and by
expression of DNAs in cells and organisms.
[0078] The term "diagnosis" is used herein to refer to the
identification or classification of a molecular or pathological
state, disease or condition (e.g., cancer). For example,
"diagnosis" may refer to identification of a particular type of
cancer. "Diagnosis" may also refer to the classification of a
particular subtype of cancer, e.g., by histopathological criteria,
or by molecular features (e.g., a subtype characterized by
expression of one or a combination of biomarkers (e.g., particular
genes or proteins encoded by said genes)).
[0079] The term "sample," as used herein, refers to a composition
that is obtained or derived from a subject and/or individual of
interest that contains a cellular and/or other molecular entity
that is to be characterized and/or identified, for example based on
physical, biochemical, chemical and/or physiological
characteristics. For example, the phrase "disease sample" and
variations thereof refers to any sample obtained from a subject of
interest that would be expected or is known to contain the cellular
and/or molecular entity that is to be characterized. Samples
include, but are not limited to, primary or cultured cells or cell
lines, cell supernatants, cell lysates, platelets, serum, plasma,
vitreous fluid, lymph fluid, synovial fluid, follicular fluid,
seminal fluid, amniotic fluid, milk, whole blood, blood-derived
cells, urine, cerebro-spinal fluid, saliva, sputum, tears,
perspiration, mucus, tumor lysates, and tissue culture medium,
tissue extracts such as homogenized tissue, tumor tissue, cellular
extracts, and combinations thereof.
[0080] By "tissue sample" or "cell sample" is meant a collection of
similar cells obtained from a tissue of a subject or individual.
The source of the tissue or cell sample may be solid tissue as from
a fresh, frozen and/or preserved organ, tissue sample, biopsy,
and/or aspirate; blood or any blood constituents such as plasma;
bodily fluids such as cerebral spinal fluid, amniotic fluid,
peritoneal fluid, or interstitial fluid; cells from any time in
gestation or development of the subject. The tissue sample may also
be primary or cultured cells or cell lines. Optionally, the tissue
or cell sample is obtained from a disease tissue/organ. The tissue
sample may contain compounds which are not naturally intermixed
with the tissue in nature such as preservatives, anticoagulants,
buffers, fixatives, nutrients, antibiotics, or the like.
[0081] A "reference sample", "reference cell", "reference tissue",
"control sample", "control cell", or "control tissue", as used
herein, refers to a sample, cell, tissue, standard, or level that
is used for comparison purposes. In one embodiment, a reference
sample, reference cell, reference tissue, control sample, control
cell, or control tissue is obtained from a healthy and/or
non-diseased part of the body (e.g., tissue or cells) of the same
subject or individual. For example, healthy and/or non-diseased
cells or tissue adjacent to the diseased cells or tissue (e.g.,
cells or tissue adjacent to a tumor). In another embodiment, a
reference sample is obtained from an untreated tissue and/or cell
of the body of the same subject or individual. In yet another
embodiment, a reference sample, reference cell, reference tissue,
control sample, control cell, or control tissue is obtained from a
healthy and/or non-diseased part of the body (e.g., tissues or
cells) of an individual who is not the subject or individual. In
even another embodiment, a reference sample, reference cell,
reference tissue, control sample, control cell, or control tissue
is obtained from an untreated tissue and/or cell of the body of an
individual who is not the subject or individual.
[0082] For the purposes herein a "section" of a tissue sample is
meant a single part or piece of a tissue sample, e.g. a thin slice
of tissue or cells cut from a tissue sample. It is understood that
multiple sections of tissue samples may be taken and subjected to
analysis, provided that it is understood that the same section of
tissue sample may be analyzed at both morphological and molecular
levels, or analyzed with respect to both polypeptides and
polynucleotides.
[0083] By "correlate" or "correlating" is meant comparing, in any
way, the performance and/or results of a first analysis or protocol
with the performance and/or results of a second analysis or
protocol. For example, one may use the results of a first analysis
or protocol in carrying out a second protocols and/or one may use
the results of a first analysis or protocol to determine whether a
second analysis or protocol should be performed. With respect to
the embodiment of polypeptide analysis or protocol, one may use the
results of the polypeptide expression analysis or protocol to
determine whether a specific therapeutic regimen should be
performed. With respect to the embodiment of polynucleotide
analysis or protocol, one may use the results of the polynucleotide
expression analysis or protocol to determine whether a specific
therapeutic regimen should be performed.
[0084] "Individual response" or "response" can be assessed using
any endpoint indicating a benefit to the individual, including,
without limitation, (1) inhibition, to some extent, of disease
progression (e.g., cancer progression), including slowing down and
complete arrest; (2) a reduction in tumor size; (3) inhibition
(i.e., reduction, slowing down or complete stopping) of cancer cell
infiltration into adjacent peripheral organs and/or tissues; (4)
inhibition (i.e. reduction, slowing down or complete stopping) of
metatasis; (5) relief, to some extent, of one or more symptoms
associated with the disease or disorder (e.g., cancer); (6)
increase or extend in the length of survival, including overall
survival and progression free survival; and/or (7) decreased
mortality at a given point of time following treatment.
[0085] An "effective response" of a patient or a patient's
"responsiveness" to treatment with a medicament and similar wording
refers to the clinical or therapeutic benefit imparted to a patient
at risk for, or suffering from, a disease or disorder, such as
cancer. In one embodiment, such benefit includes any one or more
of: extending survival (including overall survival and progression
free survival); resulting in an objective response (including a
complete response or a partial response); or improving signs or
symptoms of cancer. In one embodiment, the presence of the
biomarker is used to identify a patient who is more likely to
respond to treatment with a medicament, relative to a patient that
does not have the presence of the biomarker. In another embodiment,
the presence of the biomarker is used to determine that a patient
will have an increase likelihood of benefit from treatment with a
medicament, relative to a patient that does not have the presence
of the biomarker.
[0086] "Survival" refers to the patient remaining alive, and
includes overall survival as well as progression free survival.
[0087] "Overall survival" refers to the patient remaining alive for
a defined period of time, such as 1 year, 5 years, etc from the
time of diagnosis or treatment.
[0088] "Progression free survival" refers to the patient remaining
alive, without the cancer progressing or getting worse.
[0089] By "extending survival" is meant increasing overall or
progression free survival in a treated patient relative to an
untreated patient (i.e. relative to a patient not treated with the
medicament), or relative to a patient who does not express a
biomarker at the designated level, and/or relative to a patient
treated with an approved anti-tumor agent. An objective response
refers to a measurable response, including complete response (CR)
or partial response (PR).
[0090] By complete response or "CR" is intended the disappearance
of all signs of cancer in response to treatment. This does not
always mean the cancer has been cured.
[0091] Partial response or "PR" refers to a decrease in the size of
one or more tumors or lesions, or in the extent of cancer in the
body, in response to treatment.
[0092] An "effective amount" of an agent refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired therapeutic or prophylactic result.
[0093] A "therapeutically effective amount" refers to an amount of
a therapeutic agent to treat or prevent a disease or disorder in a
mammal. In the case of cancers, the therapeutically effective
amount of the therapeutic agent may reduce the number of cancer
cells; reduce the primary tumor size; inhibit (i.e., slow to some
extent and preferably stop) cancer cell infiltration into
peripheral organs; inhibit (i.e., slow to some extent and
preferably stop) tumor metastasis; inhibit, to some extent, tumor
growth; and/or relieve to some extent one or more of the symptoms
associated with the disorder. To the extent the drug may prevent
growth and/or kill existing cancer cells, it may be cytostatic
and/or cytotoxic. For cancer therapy, efficacy in vivo can, for
example, be measured by assessing the duration of survival, time to
disease progression (TTP), the response rates (RR), duration of
response, and/or quality of life.
[0094] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. Included in this definition are benign
and malignant cancers. By "early stage cancer" or "early stage
tumor" is meant a cancer that is not invasive or metastatic or is
classified as a Stage 0, I, or II cancer. Examples of cancer
include, but are not limited to, carcinoma, lymphoma, blastoma
(including medulloblastoma and retinoblastoma), sarcoma (including
liposarcoma and synovial cell sarcoma), neuroendocrine tumors
(including carcinoid tumors, gastrinoma, and islet cell cancer),
mesothelioma, schwannoma (including acoustic neuroma), meningioma,
adenocarcinoma, melanoma, and leukemia or lymphoid malignancies.
More particular examples of such cancers include squamous cell
cancer (e.g. epithelial squamous cell cancer), lung cancer
including small-cell lung cancer (SCLC), non-small cell lung cancer
(NSCLC), adenocarcinoma of the lung and squamous carcinoma of the
lung, cancer of the peritoneum, hepatocellular cancer, gastric or
stomach cancer including gastrointestinal cancer, pancreatic
cancer, glioblastoma, cervical cancer, ovarian cancer, liver
cancer, bladder cancer, hepatoma, breast cancer (including
metastatic breast cancer), colon cancer, rectal cancer, colorectal
cancer, endometrial or uterine carcinoma, salivary gland carcinoma,
kidney or renal cancer, prostate cancer, vulval cancer, thyroid
cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, merkel
cell cancer, mycoses fungoids, testicular cancer, esophageal
cancer, tumors of the biliary tract, as well as head and neck
cancer and hematological malignancies. In some embodiments, the
cancer is triple-negative metastatic breast cancer, including any
histologically confirmed triple-negative (ER-, PR-, HER2-)
adenocarcinoma of the breast with locally recurrent or metastatic
disease (where the locally recurrent disease is not amenable to
resection with curative intent).
[0095] The term "pharmaceutical formulation" refers to a
preparation which is in such form as to permit the biological
activity of an active ingredient contained therein to be effective,
and which contains no additional components which are unacceptably
toxic to a subject to which the formulation would be
administered.
[0096] A "pharmaceutically acceptable carrier" refers to an
ingredient in a pharmaceutical formulation, other than an active
ingredient, which is nontoxic to a subject., A pharmaceutically
acceptable carrier includes, but is not limited to, a buffer,
excipient, stabilizer, or preservative.
[0097] As used herein, "treatment" (and grammatical variations
thereof such as "treat" or "treating") refers to clinical
intervention in an attempt to alter the natural course of the
individual being treated, and can be performed either for
prophylaxis or during the course of clinical pathology. Desirable
effects of treatment include, but are not limited to, preventing
occurrence or recurrence of disease, alleviation of symptoms,
diminishment of any direct or indirect pathological consequences of
the disease, preventing metastasis, decreasing the rate of disease
progression, amelioration or palliation of the disease state, and
remission or improved prognosis. In some embodiments, antibodies
are used to delay development of a disease or to slow the
progression of a disease.
[0098] The term "anti-cancer therapy" refers to a therapy useful in
treating cancer. Examples of anti-cancer therapeutic agents
include, but are limited to, e.g., chemotherapeutic agents, growth
inhibitory agents, cytotoxic agents, agents used in radiation
therapy, anti-angiogenesis agents, apoptotic agents, anti-tubulin
agents, and other agents to treat cancer, anti-CD20 antibodies,
platelet derived growth factor inhibitors (e.g., Gleevec.TM.
(Imatinib Mesylate)), a COX-2 inhibitor (e.g., celecoxib),
interferons, cytokines, antagonists (e.g., neutralizing antibodies)
that bind to one or more of the following targets PDGFR-beta, BlyS,
APRIL, BCMA receptor(s), TRAIL/Apo2, and other bioactive and
organic chemical agents, etc. Combinations thereof are also
included in the invention.
[0099] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188,
Sm153, Bi212, P32 and radioactive isotopes of Lu), chemotherapeutic
agents e.g., methotrexate, adriamicin, vinca alkaloids
(vincristine, vinblastine, etoposide), doxorubicin, melphalan,
mitomycin C, chlorambucil, daunorubicin or other intercalating
agents, enzymes and fragments thereof such as nucleolytic enzymes,
antibiotics, and toxins such as small molecule toxins or
enzymatically active toxins of bacterial, fungal, plant or animal
origin, including fragments and/or variants thereof, and the
various antitumor or anticancer agents disclosed below. Other
cytotoxic agents are described below. A tumoricidal agent causes
destruction of tumor cells.
[0100] A "chemotherapeutic agent" refers to a chemical compound
useful in the treatment of cancer. Examples of chemotherapeutic
agents include alkylating agents such as thiotepa and
cyclosphosphamide (CYTOXAN.RTM.); alkyl sulfonates such as
busulfan, improsulfan and piposulfan; aziridines such as benzodopa,
carboquone, meturedopa, and uredopa; ethylenimines and
methylamelamines including altretamine, triethylenemelamine,
triethylenephosphoramide, triethylenethiophosphoramide and
trimethylomelamine; acetogenins (especially bullatacin and
bullatacinone); delta-9-tetrahydrocannabinol (dronabinol,
MARINOL.RTM.); beta-lapachone; lapachol; colchicines; betulinic
acid; a camptothecin (including the synthetic analogue topotecan
(HYCAMTIN.RTM.), CPT-11 (irinotecan, CAMPTOSAR.RTM.),
acetylcamptothecin, scopolectin, and 9-aminocamptothecin);
bryostatin; callystatin; CC-1065 (including its adozelesin,
carzelesin and bizelesin synthetic analogues); podophyllotoxin;
podophyllinic acid; teniposide; cryptophycins (particularly
cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
(including the synthetic analogues, KW-2189 and CB1-TM1);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin;
nitrogen mustards such as chlorambucil, chlornaphazine,
chlorophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosoureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, and ranimnustine; antibiotics such as the
enediyne antibiotics (e.g., calicheamicin, especially calicheamicin
gamma1I and calicheamicin omegaI1 (see, e.g., Nicolaou et al.,
Angew. Chem Intl. Ed. Engl., 33: 183-186 (1994)); CDP323, an oral
alpha-4 integrin inhibitor; dynemicin, including dynemicin A; an
esperamicin; as well as neocarzinostatin chromophore and related
chromoprotein enediyne antibiotic chromophores), aclacinomysins,
actinomycin, authramycin, azaserine, bleomycins, cactinomycin,
carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin
(including ADRIAMYCIN.RTM., morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin
HCl liposome injection (DOXIL.RTM.), liposomal doxorubicin TLC D-99
(MYOCET.RTM.), peglylated liposomal doxorubicin (CAELYX.RTM.), and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate, gemcitabine (GEMZAR.RTM.), tegafur (UFTORAL.RTM.),
capecitabine (XELODA.RTM.), an epothilone, and 5-fluorouracil
(5-FU); folic acid analogues such as denopterin, methotrexate,
pteropterin, trimetrexate; purine analogs such as fludarabine,
6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such
as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine,
dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens
such as calusterone, dromostanolone propionate, epitiostanol,
mepitiostane, testolactone; anti-adrenals such as
aminoglutethimide, mitotane, trilostane; folic acid replenisher
such as frolinic acid; aceglatone; aldophosphamide glycoside;
aminolevulinic acid; eniluracil; amsacrine; bestrabucil;
bisantrene; edatraxate; defofamine; demecolcine; diaziquone;
elfornithine; elliptinium acetate; an epothilone; etoglucid;
gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids
such as maytansine and ansamitocins; mitoguazone; mitoxantrone;
mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin;
losoxantrone; 2-ethylhydrazide; procarbazine; PSK.RTM.
polysaccharide complex (JHS Natural Products, Eugene, Oreg.);
razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid;
triaziquone; 2,2',2'-trichlorotriethylamine; trichothecenes
(especially T-2 toxin, verracurin A, roridin A and anguidine);
urethan; vindesine (ELDISINE.RTM., FILDESIN.RTM.)); dacarbazine;
mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;
arabinoside ("Ara-C"); thiotepa; taxoid, e.g., paclitaxel
(TAXOL.RTM.), albumin-engineered nanoparticle formulation of
paclitaxel (ABRAXANE.TM.), and docetaxel (TAXOTERE.RTM.);
chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum
agents such as cisplatin, oxaliplatin (e.g., ELOXATIN.RTM.), and
carboplatin; vincas, which prevent tubulin polymerization from
forming microtubules, including vinblastine (VELBAN.RTM.),
vincristine (ONCOVIN.RTM.), vindesine (ELDISINE.RTM.,
FILDESIN.RTM.), and vinorelbine (NAVELBINE.RTM.); etoposide
(VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone;
edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase
inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such
as retinoic acid, including bexarotene (TARGRETIN.RTM.);
bisphosphonates such as clodronate (for example, BONEFOS.RTM. or
OSTAC.RTM.), etidronate (DIDROCAL.RTM.), NE-58095, zoledronic
acid/zoledronate (ZOMETA.RTM.), alendronate (FOSAMAX.RTM.),
pamidronate (AREDIA.RTM.), tiludronate (SKELID.RTM.), or
risedronate (ACTONEL.RTM.); troxacitabine (a 1,3-dioxolane
nucleoside cytosine analog); antisense oligonucleotides,
particularly those that inhibit expression of genes in signaling
pathways implicated in aberrant cell proliferation, such as, for
example, PKC-alpha, Raf, H-Ras, and epidermal growth factor
receptor (EGF-R); vaccines such as THERATOPE.RTM. vaccine and gene
therapy vaccines, for example, ALLOVECTIN.RTM. vaccine,
LEUVECTIN.RTM. vaccine, and VAXID.RTM. vaccine; topoisomerase 1
inhibitor (e.g., LURTOTECAN.RTM.); rmRH (e.g., ABARELIX.RTM.);
BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT.RTM.,
Pfizer); perifosine, COX-2 inhibitor (e.g., celecoxib or
etoricoxib), proteosome inhibitor (e.g., PS341); bortezomib
(VELCADE.RTM.); CCI-779; tipifarnib (R11577); orafenib, ABT510;
Bcl-2 inhibitor such as oblimersen sodium (GENASENSE.RTM.);
pixantrone; EGFR inhibitors (see definition below); tyrosine kinase
inhibitors (see definition below); serine-threonine kinase
inhibitors such as rapamycin (sirolimus, RAPAMUNE.RTM.);
farnesyltransferase inhibitors such as lonafarnib (SCH 6636,
SARASAR.TM.); and pharmaceutically acceptable salts, acids or
derivatives of any of the above; as well as combinations of two or
more of the above such as CHOP, an abbreviation for a combined
therapy of cyclophosphamide, doxorubicin, vincristine, and
prednisolone; and FOLFOX, an abbreviation for a treatment regimen
with oxaliplatin (ELOXATIN.TM.) combined with 5-FU and
leucovorin.
[0101] Chemotherapeutic agents as defined herein include
"anti-hormonal agents" or "endocrine therapeutics" which act to
regulate, reduce, block, or inhibit the effects of hormones that
can promote the growth of cancer. They may be hormones themselves,
including, but not limited to: anti-estrogens with mixed
agonist/antagonist profile, including, tamoxifen (NOLVADEX.RTM.),
4-hydroxytamoxifen, toremifene (FARESTON.RTM.), idoxifene,
droloxifene, raloxifene (EVISTA.RTM.), trioxifene, keoxifene, and
selective estrogen receptor modulators (SERMs) such as SERM3; pure
anti-estrogens without agonist properties, such as fulvestrant
(FASLODEX.RTM.), and EM800 (such agents may block estrogen receptor
(ER) dimerization, inhibit DNA binding, increase ER turnover,
and/or suppress ER levels); aromatase inhibitors, including
steroidal aromatase inhibitors such as formestane and exemestane
(AROMASIN.RTM.), and nonsteroidal aromatase inhibitors such as
anastrazole (ARIMIDEX.RTM.), letrozole (FEMARA.RTM.) and
aminoglutethimide, and other aromatase inhibitors include vorozole
(RIVISOR.RTM.), megestrol acetate (MEGASE.RTM.), fadrozole, and
4(5)-imidazoles; lutenizing hormone-releaseing hormone agonists,
including leuprolide (LUPRON.RTM. and ELIGARD.RTM.), goserelin,
buserelin, and tripterelin; sex steroids, including progestines
such as megestrol acetate and medroxyprogesterone acetate,
estrogens such as diethylstilbestrol and premarin, and
androgens/retinoids such as fluoxymesterone, all transretionic acid
and fenretinide; onapristone; anti-progesterones; estrogen receptor
down-regulators (ERDs); anti-androgens such as flutamide,
nilutamide and bicalutamide; and pharmaceutically acceptable salts,
acids or derivatives of any of the above; as well as combinations
of two or more of the above.
[0102] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell (e.g., a
cell whose growth is dependent upon PD-L1 expression either in
vitro or in vivo). Examples of growth inhibitory agents include
agents that block cell cycle progression (at a place other than S
phase), such as agents that induce G1 arrest and M-phase arrest.
Classical M-phase blockers include the vincas (vincristine and
vinblastine), taxanes, and topoisomerase II inhibitors such as
doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
Those agents that arrest G1 also spill over into S-phase arrest,
for example, DNA alkylating agents such as tamoxifen, prednisone,
dacarbazine, mechlorethamine, cisplatin, methotrexate,
5-fluorouracil, and ara-C. Further information can be found in The
Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1,
entitled "Cell cycle regulation, oncogenes, and antineoplastic
drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995),
especially p. 13. The taxanes (paclitaxel and docetaxel) are
anticancer drugs both derived from the yew tree. Docetaxel
(TAXOTERE.RTM., Rhone-Poulenc Rorer), derived from the European
yew, is a semisynthetic analogue of paclitaxel (TAXOL.RTM.,
Bristol-Myers Squibb). Paclitaxel and docetaxel promote the
assembly of microtubules from tubulin dimers and stabilize
microtubules by preventing depolymerization, which results in the
inhibition of mitosis in cells.
[0103] By "radiation therapy" is meant the use of directed gamma
rays or beta rays to induce sufficient damage to a cell so as to
limit its ability to function normally or to destroy the cell
altogether. It will be appreciated that there will be many ways
known in the art to determine the dosage and duration of treatment.
Typical treatments are given as a one time administration and
typical dosages range from 10 to 200 units (Grays) per day.
[0104] An "individual" or "subject" is a mammal. Mammals include,
but are not limited to, domesticated animals (e.g., cows, sheep,
cats, dogs, and horses), primates (e.g., humans and non-human
primates such as monkeys), rabbits, and rodents (e.g., mice and
rats). In certain embodiments, the individual or subject is a
human.
[0105] The term "concurrently" is used herein to refer to
administration of two or more therapeutic agents, where at least
part of the administration overlaps in time. Accordingly,
concurrent administration includes a dosing regimen when the
administration of one or more agent(s) continues after
discontinuing the administration of one or more other agent(s).
[0106] By "reduce or inhibit" is meant the ability to cause an
overall decrease of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%,
90%, 95%, or greater. Reduce or inhibit can refer to the symptoms
of the disorder being treated, the presence or size of metastases,
or the size of the primary tumor.
[0107] It is understood that the singular form "a", "an", and "the"
includes plural references unless indicated otherwise.
[0108] Dendritic Cell Markers
[0109] In the present invention, abundance of DCs in a tumor tissue
sample obtained from a patient with cancer (i.e. tumor-infiltrating
DCs), more preferably functional DCs with cross-presenting
capacity, was found to be predictive of response to PD-1 axis
inhibitors. Abundance of DCs can be determined by detecting
expression levels of markers associated with development,
activation or manuration of DCs with cross-presenting properties.
Those markers include XCR1, IRF8, BATF3, FLT3. These markers may be
considered separately, as individual markers, or in groups of two
or more markers, as a cumulative expression of the markers, i.e. a
cumulative DC gene score (DC score). The expression levels of two
or more markers can be combined by any appropriate state of the art
mathematical method to obtain a DC score. In one embodiment, a DC
score can be obtained on the basis of expression levels of genes
consisting of XCR1, IRF8, BATF3, and FLT3.
[0110] In one embodiment, the biomarker of the present invention is
used for predicting response of patients with renal cell carcinoma
to a PD-1 axis inhibitor such as an anti-PD-L1 antibody
atezolizumab. In another embodiment, the biomarker of the present
invention is used for predicting response of patients with
non-small cell lung cancer (NSCLC) to a PD-1 axis inhibitor such as
an anti-PD-L1 antibody atezolizumab. According to the embodiments
of the present invention, the predictive value of the present
invention is higher in patients who are PD-L1 positive, and in
patients with squamous NSCLC. Therefore, in one embodiment, the
biomarker of the present invention is used for predicting response
of patients who are PD-L1 positive, more specifically patients with
NSCLC who are PD-L1 positive, to a PD-1 axis inhibitor such as an
anti-PD-L1 antibody atezolizumab. In another embodiment, the
biomarker of the present invention is used for predicting response
of patients with squamous NSCLC to a PD-1 axis inhibitor such as an
anti-PD-L1 antibody atezolizumab.
[0111] Exemplary PD-1 Axis Inhibitors for Use in the Present
Invention By way of example, a PD-1 axis inhibitor includes a PD-1
binding antagonist and a PD-L1 binding antagonist. Alternative
names for "PD-1" include CD279 and SLEB2. Alternative names for
"PD-L1" include B7-H1, B7-4, CD274, and B7-H. In some embodiments,
PD-1 and PD-L1 are human PD-1 and PD-L1.
[0112] In some embodiments, the PD-1 binding antagonist is a
molecule that inhibits the binding of PD-1 to its ligand binding
partners. In a specific aspect the PD-1 ligand binding partners are
PD-L1 and/or PDL2. In another embodiment, a PDL1 binding antagonist
is a molecule that inhibits the binding of PD-L1 to its binding
partners. In a specific aspect, PD-L1 binding partners are PD-1
and/or B7-1. The antagonist may be an antibody, an antigen binding
fragment thereof, an immunoadhesin, a fusion protein, or
oligopeptide.
[0113] In some embodiments, the PD-1 binding antagonist is an
anti-PD-1 antibody (e.g., a human antibody, a humanized antibody,
or a chimeric antibody). In some embodiments, the anti-PD-1
antibody is selected from the group consisting of nivolumab and
pembrolizumab. In some embodiments, the PD-1 binding antagonist is
an immunoadhesin (e.g., an immunoadhesin comprising an
extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a
constant region (e.g., an Fc region of an immunoglobulin sequence).
In some embodiments, the PD-1 binding antagonist is AMP-224.
Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538,
BMS-936558, and OPDIVO.RTM., is an anti-PD-1 antibody described in
WO2006/121168. Pembrolizumab, also known as MK-3475, Merck 3475,
lambrolizumab, KEYTRUDA.RTM., and SCH-900475, is an anti-PD-1
antibody described in WO2009/114335. AMP-224, also known as
B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in
WO2010/027827 and WO2011/066342.
[0114] In some embodiments, the PD-L1 binding antagonist is
anti-PD-L1 antibody. In some embodiments, the anti-PDL1 binding
antagonist is selected from the group consisting of YW243.55.570,
MPDL3280A, MDX-1105, and MEDI4736. MDX-1105, also known as
BMS-936559, is an anti-PDL1 antibody described in WO2007/005874.
Antibody YW243.55.570 is an anti-PDL1 described in WO 2010/077634
A1. MEDI4736 is an anti-PDL1 antibody described in WO2011/066389
and US2013/034559.
[0115] Examples of anti-PD-L1 antibodies useful for the methods of
this invention, and methods for making thereof are described in PCT
patent application WO 2010/077634 A1 and U.S. Pat. No. 8,217,149,
each incorporated herein by reference as if set forth in their
entirety.
[0116] In some embodiments, the anti-PD-L1 antibody is atezolizumab
(CAS Registry Number: 1422185-06-5). Atezolizumab (Genentech), also
known as MPDL3280A, is an anti-PD-L1 antibody.
[0117] Atezolizumab comprises:
[0118] (a) an HVR-H1, HVR-H2, and HVR-H3 sequence of GFTFSDSWIH
(SEQ ID NO:1), AWISPYGGSTYYADSVKG (SEQ ID NO:2) and RHWPGGFDY (SEQ
ID NO:3), respectively, and
[0119] (b) an HVR-L1, HVR-L2, and HVR-L3 sequence of RASQDVSTAVA
(SEQ ID NO:4), SASFLYS (SEQ ID NO:5) and QQYLYHPAT (SEQ ID NO:6),
respectively.
[0120] Atezolizumab comprises a heavy chain and a light chain
sequence, wherein:
TABLE-US-00001 (a) the heavy chain variable region sequence
comprises the amino acid sequence: (SEQ ID NO: 7)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAW
ISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRH
WPGGFDYWGQGTLVTVSS, and (b) the light chain variable region
sequence comprises the amino acid sequence: (SEQ ID NO: 8)
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYS
ASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQ GTKVEIKR.
[0121] Atezolizumab comprises a heavy chain and a light chain
sequence, wherein:
TABLE-US-00002 (a) the heavy chain comprises the amino acid
sequence: (SEQ ID NO: 9)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAW
ISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRH
WPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY
FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI
CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD
TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYAST
YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY
TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, and (b) the light
chain comprises the amino acid sequence: (SEQ ID NO: 10)
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYS
ASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC.
[0122] In some embodiments, the PD-1 axis binding antagonist is an
anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody
is capable of inhibiting binding between PD-L1 and PD-1 and/or
between PD-L1 and B7-1. In some embodiments, the anti-PD-L1
antibody is a monoclonal antibody. In some embodiments, the
anti-PD-L1 antibody is an antibody fragment selected from the group
consisting of Fab, Fab'-SH, Fv, scFv, and (Fab')2 fragments. In
some embodiments, the anti-PDL1 antibody is a humanized antibody.
In some embodiments, the anti-PDL1 antibody is a human
antibody.
[0123] The antibody or antigen binding fragment thereof, may be
made using methods known in the art, for example, by a process
comprising culturing a host cell containing nucleic acid encoding
any of the previously described anti-PD-L1, anti-PD-1, or
anti-PD-L2 antibodies or antigen-binding fragment in a form
suitable for expression, under conditions suitable to produce such
antibody or fragment, and recovering the antibody or fragment.
[0124] In any of the embodiments herein, the isolated anti-PDL1
antibody can bind to a human PDL1, for example a human PDL1 as
shown in UniProtKB/Swiss-Prot Accession No.Q9NZQ7.1, or a variant
thereof.
[0125] In a still further embodiment, the invention provides for a
composition comprising an anti-PD-L1, an anti-PD-1, or an
anti-PD-L2 antibody or antigen binding fragment thereof as provided
herein and at least one pharmaceutically acceptable carrier. In
some embodiments, the anti-PD-L1, anti-PD-1, or anti-PD-L2 antibody
or antigen binding fragment thereof administered to the individual
is a composition comprising one or more pharmaceutically acceptable
carrier.
[0126] In some embodiments, the anti-PD-L1 antibody described
herein is in a formulation comprising the antibody at an amount of
about 60 mg/mL, histidine acetate in a concentration of about 20
mM, sucrose in a concentration of about 120 mM, and polysorbate
(e.g., polysorbate 20) in a concentration of 0.04% (w/v), and the
formulation has a pH of about 5.8. In some embodiments, the
anti-PD-L1 antibody described herein is in a formulation comprising
the antibody in an amount of about 125 mg/mL, histidine acetate in
a concentration of about 20 mM, sucrose is in a concentration of
about 240 mM, and polysorbate (e.g., polysorbate 20) in a
concentration of 0.02% (w/v), and the formulation has a pH of about
5.5.
[0127] Assays for Use in the Present Invention
[0128] In some embodiments, the biomarker is detected in the sample
using a method selected from the group consisting of FACS, Western
blot, ELISA, immunoprecipitation, immunohistochemistry,
immunofluorescence, radioimmunoassay, immunodetection methods, mass
spectrometery, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq,
microarray analysis, nanostring, SAGE, MassARRAY technique, and
FISH, and combinations thereof. In some embodiments, the biomarker
is detected in the sample by protein expression. In some
embodiments, protein expression is determined by
immunohistochemistry (IHC).
[0129] In some embodiments, the biomarker is detected in the sample
by mRNA expression. In some embodiments, the mRNA expression is
determined using qPCR, rtPCR, RNA-seq, multiplex qPCR or RT-qPCR,
microarray analysis, nanostring, SAGE, MassARRAY technique, or
FISH.
[0130] In some embodiments, the sample is a tumor tissue sample. In
some embodiments, the tumor tissue sample comprises tumor cells,
tumor infiltrating immune cells, stromal cells or any combinations
thereof.
[0131] In some embodiments, the sample is obtained prior to
treatment with a PD-L1 axis inhibitor. In some embodiments, the
tissue sample is formalin fixed and paraffin embedded, archival,
fresh or frozen.
[0132] Presence and/or expression level/amount of various
biomarkers in a sample can be analyzed by a number of
methodologies, many of which are known in the art and understood by
the skilled artisan, including, but not limited to,
immunohistochemistry ("IHC"), Western blot analysis,
immunoprecipitation, molecular binding assays, ELISA, ELIFA,
fluorescence activated cell sorting ("FACS"), MassARRAY,
proteomics, quantitative blood based assays (as for example Serum
ELISA), biochemical enzymatic activity assays, in situ
hybridization, Southern analysis, Northern analysis, whole genome
sequencing, polymerase chain reaction ("PCR") including
quantitative real time PCR ("qRT-PCR") and other amplification type
detection methods, such as, for example, branched DNA, SISBA, TMA
and the like), RNA-Seq, FISH, microarray analysis, gene expression
profiling, and/or serial analysis of gene expression ("SAGE"), as
well as any one of the wide variety of assays that can be performed
by protein, gene, and/or tissue array analysis. Typical protocols
for evaluating the status of genes and gene products are found, for
example in Ausubel et al., eds., 1995, Current Protocols In
Molecular Biology, Units 2 (Northern Blotting), 4 (Southern
Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Multiplexed
immunoassays such as those available from Rules Based Medicine or
Meso Scale Discovery ("MSD") may also be used.
[0133] In one embodiment, the sample is a clinical sample. In
another embodiment, the sample is used in a diagnostic assay. In
some embodiments, the sample is obtained from a primary or
metastatic tumor. Tissue biopsy is often used to obtain a
representative piece of tumor tissue.
[0134] In certain embodiments, a reference sample, reference
tissue, control sample, or control tissue is a single sample or
combined multiple samples from the same subject or individual that
are obtained at one or more different time points than when the
test sample is obtained. In certain embodiments, a reference
sample, reference tissue, control sample, or control tissue is a
combined multiple samples from one or more healthy individuals who
are not the subject or individual. In certain embodiments, a
reference sample, reference tissue, control sample, or control
tissue is a combined multiple samples from one or more individuals
with a disease or disorder (e.g., cancer) who are not the subject
or individual.
[0135] In some embodiments, the sample is a tumor tissue sample
(e.g., biopsy tissue). In some embodiments, the tissue sample is
lung tissue. In some embodiments, the tissue sample is renal
tissue. In some embodiments, the tissue sample is skin tissue. In
some embodiments, the tissue sample is pancreatic tissue. In some
embodiments, the tissue sample is gastric tissue. In some
embodiments, the tissue sample is bladder tissue. In some
embodiments, the tissue sample is esophageal tissue. In some
embodiments, the tissue sample is mesothelial tissue. In some
embodiments, the tissue sample is breast tissue. In some
embodiments, the tissue sample is thyroid tissue. In some
embodiments, the tissue sample is colorectal tissue. In some
embodiments, the tissue sample is head and neck tissue. In some
embodiments, the tissue sample is osteosarcoma tissue. In some
embodiments, the tissue sample is prostate tissue. In some
embodiments, the tissue sample is ovarian tissue, HCC (liver),
blood cells, lymph nodes, bone/bone marrow.
[0136] Therapeutic Methods
[0137] Provided are methods for treating cancer in an individual,
the method comprising: determining the abundance of DCs in a tumor
tissue sample from the individual, and administering an effective
amount of a PD-1 axis inhibitor to the individual.
[0138] In some embodiments, an increased expression of biomarkers
related to development of DCs indicates that the individual is more
likely to have increased clinical benefit when the individual is
treated with the PD-L1 axis inhibitor. In some embodiments, the
increased clinical benefit comprises a relative increase in one or
more of the following: overall survival (OS), progression free
survival (PFS), complete response (CR), partial response (PR) and
combinations thereof.
[0139] PD-1 axis inhibitor described herein can be used either
alone or in combination with other agents in a therapy. For
instance, a PD-1 axis inhibitor described herein may be
co-administered with at least one additional therapeutic agent. In
certain embodiments, an additional therapeutic agent is a
chemotherapeutic agent.
[0140] Such combination therapies noted above encompass combined
administration (where two or more therapeutic agents are included
in the same or separate formulations), and separate administration,
in which case, administration of the antagonist can occur prior to,
simultaneously, and/or following, administration of the additional
therapeutic agent and/or adjuvant. PD-1 axis inhibitor described
herein can also be used in combination with radiation therapy.
[0141] A PD-1 axis inhibitor (e.g., an antibody, binding
polypeptide, and/or small molecule) described herein (and any
additional therapeutic agent) can be administered by any suitable
means, including parenteral, intrapulmonary, and intranasal, and,
if desired for local treatment, intralesional administration.
Parenteral infusions include intramuscular, intravenous,
intraarterial, intraperitoneal, or subcutaneous administration.
Dosing can be by any suitable route, e.g., by injections, such as
intravenous or subcutaneous injections, depending in part on
whether the administration is brief or chronic. Various dosing
schedules including but not limited to single or multiple
administrations over various time-points, bolus administration, and
pulse infusion are contemplated herein.
[0142] PD-1 axis inhibitor (e.g., an antibody, binding polypeptide,
and/or small molecule) described herein may be formulated, dosed,
and administered in a fashion consistent with good medical
practice. Factors for consideration in this context include the
particular disorder being treated, the particular mammal being
treated, the clinical condition of the individual patient, the
cause of the disorder, the site of delivery of the agent, the
method of administration, the scheduling of administration, and
other factors known to medical practitioners. The PD-1 axis
inhibitor need not be, but is optionally formulated with one or
more agents currently used to prevent or treat the disorder in
question. The effective amount of such other agents depends on the
amount of the PD-1 axis inhibitor present in the formulation, the
type of disorder or treatment, and other factors discussed above.
These are generally used in the same dosages and with
administration routes as described herein, or about from 1 to 99%
of the dosages described herein, or in any dosage and by any route
that is empirically/clinically determined to be appropriate.
[0143] For the prevention or treatment of disease, the appropriate
dosage of a PD-1 axis inhibitor described herein (when used alone
or in combination with one or more other additional therapeutic
agents) will depend on the type of disease to be treated, the
severity and course of the disease, whether the PD-1 axis inhibitor
is administered for preventive or therapeutic purposes, previous
therapy, the patient's clinical history and response to the PD-1
axis inhibitor, and the discretion of the attending physician. The
PD-1 axis inhibitor is suitably administered to the patient at one
time or over a series of treatments. One typical daily dosage might
range from about 1 .mu.g/kg to 100 mg/kg or more, depending on the
factors mentioned above. For repeated administrations over several
days or longer, depending on the condition, the treatment would
generally be sustained until a desired suppression of disease
symptoms occurs. Such doses may be administered intermittently,
e.g., every week or every three weeks (e.g., such that the patient
receives from about two to about twenty, or e.g., about six doses
of the PD-1 axis inhibitor). An initial higher loading dose,
followed by one or more lower doses may be administered. An
exemplary dosing regimen comprises administering. However, other
dosage regimens may be useful. The progress of this therapy is
easily monitored by conventional techniques and assays.
[0144] In some embodiments, the PD-1 axis inhibitor (e.g.,
anti-PD-L1 antibody) is administered at a dosage of about 0.3-30
mg/kg. In some embodiments, the PD-L1 axis binding antagonist
(e.g., anti-PD-L1 antibody) is administered at a dosage of about
any of 0.3 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 4 mg/kg, 8 mg/kg, 15
mg/kg, 20 mg/kg, or 30 mg/kg. In some embodiments, the PD-1 axis
inhibitor (e.g., anti-PD-L1 antibody) is administered at a dosage
of about any of 2 mg/kg, 4 mg/kg, 8 mg/kg, 15 mg/kg, or 30 mg/kg in
21-day cycles. It is understood that any of the above formulations
or therapeutic methods may be carried out using an immunoconjugate
in place of or in addition to the PD-1 axis inhibitor.
[0145] Pharmaceutical formulations of a PD-1 axis inhibitor as
described herein are prepared by mixing such antibody having the
desired degree of purity with one or more optional pharmaceutically
acceptable carriers (Remington's Pharmaceutical Sciences 16th
edition, Osol, A. Ed. (1980)), in the form of lyophilized
formulations or aqueous solutions. In some embodiments, the PD-1
axis inhibitor is a binding small molecule, an antibody, binding
polypeptide, and/or polynucleotide. Pharmaceutically acceptable
carriers are generally nontoxic to recipients at the dosages and
concentrations employed, and include, but are not limited to:
buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid and methionine; preservatives
(such as octadecyldimethylbenzyl ammonium chloride; hexamethonium
chloride; benzalkonium chloride; benzethonium chloride; phenol,
butyl or benzyl alcohol; alkyl parabens such as methyl or propyl
paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and
m-cresol); low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, histidine,
arginine, or lysine; monosaccharides, disaccharides, and other
carbohydrates including glucose, mannose, or dextrins; chelating
agents such as EDTA; sugars such as sucrose, mannitol, trehalose or
sorbitol; salt-forming counter-ions such as sodium; metal complexes
(e.g., Zn-protein complexes); and/or non-ionic surfactants such as
polyethylene glycol (PEG). Exemplary pharmaceutically acceptable
carriers herein further include insterstitial drug dispersion
agents such as soluble neutral-active hyaluronidase glycoproteins
(sHASEGP), for example, human soluble PH-20 hyaluronidase
glycoproteins, such as rHuPH20 (HYLENEX.RTM., Baxter International,
Inc.). Certain exemplary sHASEGPs and methods of use, including
rHuPH20, are described in US Patent Publication Nos. 2005/0260186
and 2006/0104968. In one embodiment, a sHASEGP is combined with one
or more additional glycosaminoglycanases such as
chondroitinases.
[0146] Exemplary lyophilized formulations are described in U.S.
Pat. No. 6,267,958. Aqueous antibody formulations include those
described in U.S. Pat. No. 6,171,586 and WO2006/044908, the latter
formulations including a histidine-acetate buffer.
[0147] The formulation herein may also contain more than one active
ingredients as necessary for the particular indication being
treated, preferably those with complementary activities that do not
adversely affect each other. Such active ingredients are suitably
present in combination in amounts that are effective for the
purpose intended.
[0148] Active ingredients may be entrapped in microcapsules
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsules and poly-(methylmethacylate) microcapsules,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980).
[0149] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the PD-L1 axis
binding antagonist, which matrices are in the form of shaped
articles, e.g., films, or microcapsules.
[0150] The formulations to be used for in vivo administration are
generally sterile. Sterility may be readily accomplished, e.g., by
filtration through sterile filtration membranes.
[0151] Diagnostic Kits, Assays and Articles of Manufacture
[0152] Provided herein are diagnostic kit comprising one or more
reagent for determining the presence of a biomarker in a sample
from an individual with a disease or disorder.
[0153] Provided herein are also assay for identifying an individual
with a disease or disorder to receive a PD-L1 axis inhibitor, the
method comprising: determining the abundance of DCs in a tumor
tissue sample from the individual, and recommending a PD-1 axis
inhibitor based on the abundance of DCs.
[0154] Provided herein are also articles of manufacture comprising,
packaged together, a PD-L1 axis inhibitor (e.g., anti-PD-L1
antibodies) in a pharmaceutically acceptable carrier and a package
insert indicating that the PD-L1 axis inhibitor (e.g., anti-PD-L1
antibodies) is for treating a patient with a disease or disorder
based on abundance of DCs or expression levels biomarkers related
to development of DCs. Treatment methods include any of the
treatment methods disclosed herein. Further provided are a method
for manufacturing an article of manufacture comprising combining in
a package a pharmaceutical composition comprising a PD-1 axis
inhibitor (e.g., anti-PD-L1 antibodies) and a package insert
indicating that the pharmaceutical composition is for treating a
patient with a disease or disorder based on abundance of DCs or
expression levels of biomarkers related to development of DCs.
[0155] The article of manufacture comprises a container and a label
or package insert on or associated with the container. Suitable
containers include, for example, bottles, vials, syringes, etc. The
containers may be formed from a variety of materials such as glass
or plastic. The container holds or contains a composition
comprising the cancer medicament as the active agent and may have a
sterile access port (for example the container may be an
intravenous solution bag or a vial having a stopper pierceable by a
hypodermic injection needle).
[0156] The article of manufacture may further comprise a second
container comprising a pharmaceutically-acceptable diluent buffer,
such as bacteriostatic water for injection (BWFI),
phosphate-buffered saline, Ringer's solution and dextrose solution.
The article of manufacture may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
[0157] The article of manufacture of the present invention also
includes information, for example in the form of a package insert,
indicating that the composition is used for treating cancer based
on expression level of the biomarker(s) herein. The insert or label
may take any form, such as paper or on electronic media such as a
magnetically recorded medium (e.g., floppy disk) or a CD-ROM. The
label or insert may also include other information concerning the
pharmaceutical compositions and dosage forms in the kit or article
of manufacture.
[0158] The present invention is further described by reference to
the following non-limited figures and examples.
EXAMPLES
Example 1: PD-1 and PD-L1 are Negatively Correlated on Human
DCs
[0159] To address the potential regulation of PD-1 and its ligands
on human DCs, monocyte-derived DCs were generated using the
classical method. Briefly, Peripheral Blood Mononuclear Cells
(PBMCs) were isolated from buffy-coats (Blutspende, Schlieren) from
healthy human donors, using Ficoll-Paque.TM. Plus (GE Healthcare,
#17-1440-03) density gradient. Rings of PBMCs were collected and
washed with PBS two times by centrifugation (5' at 763 g; 5' at 600
g). A lysis was conducted using 10 mL of BD Pharm Lyse.TM. (BD
Biosciences, #555899) diluted at 1.times. with sterile water and
incubated 1.5 minute at room temperature. Two washes with
Phosphate-buffered saline 1.times. (PBS; Gibco.RTM. by live
Technologies.TM., #20012-019) were conducted by centrifugation (8'
at 135 g). Cells number and quality were evaluated using Cell
counter (Beckman coulter). Separate unrelated donors were used for
each independent experiment. Monocytes were isolated from fresh
PBMCs by negative selection using Human monocyte enrichment kit
(StemCell, #19059) in MACs buffer (1450 mL Automacs.TM. Rinsing
solution, Miltenyi #130-091-222; and 75 mL MACS BSA stock Solution,
Miltenyi #130-091-376) according to the manufacturer's
instructions. Purity was checked using Cell counter (Beckman
coulter), monocytes were routinely >95% pure.
[0160] Monocytes were seed in 6-well plates with 1.5.times.10.sup.6
cells/well in 2 mL of Medium (RPMI 1640 (1.times.) Glutamax; 10%
Heat-Inactivated Fetal Bovine Serum (FBS) and 1%
Penicillin-Streptomycin, all from Gibco.RTM.) at 37.degree. C. and
incubated 2 hours (37.degree. C., atmosphere 5% CO.sub.2). After 2
hours of incubation one more selection was processed by plastic
adherence; the medium was removed and 2 mL/well of fresh Medium
supplemented with 10 .mu.g/mL of recombinant human Interleukine-4
and Granulocyte Macrophage Colony-Stimulating Factor (IL-4 and
GM-CSF; R&D Systems, #204-IL; #215-GM) were added. Plates were
incubated at 37.degree. C. in an atmosphere with 5% CO.sub.2. On
day 2, 200 .mu.L/well of Medium at 37.degree. C. supplemented with
100 .mu.g/mL (10.times.) of recombinant human IL-4 and GM-CSF were
added. On day 4, 1 mL of Medium at 37.degree. C. supplemented with
30 .mu.g/mL (3.times.) of recombinant human IL-4 and GM-CSF were
added. After five days of culture, in vitro monocyte-derived DCs
are fully differentiated. The cells were then stained with
anti-PD-1-PE-Cy7 and anti-PD-L1-APC Abs (BioLegend; #329917,
#329707), and measured by flow cytometry (BD).
[0161] We observed that human DCs express both PD-1 and PD-L1 (FIG.
1A), the expression profile of PD-1 and PD-L1 is negatively
correlated (R.sup.2=0.907) (FIG. 1B). Upon maturation of DCs by LPS
of lipopolysaccharide (LPS, 10 ng/mL Sigma-Aldrich, #L4516), PD-1
is downregulated, while PD-L1 is upregulated (FIGS. 2A-2B), which
prompted us to hypothesize that PD-1 is a functionally negative
regulator on DCs. To test this, we generated tolerogenic DCs (tDCs,
a subset that are impaired in stimulating T cell proliferation)
following a protocol (van Kooten et al., 2011, Methods Mol Biol,
677: 149-59) by adding Dexamethasone (Sigma, #D2915) at a final
concentration of 0.39 .mu.g/mL in addition of IL-4 and GM-CSF. For
head-to-head comparison, immature DCs (iDCs), mature DCs (mDCs) and
tDCs were generated in parallel from monocytes of the same donor.
The cells were stained for the expression of PD-1, PD-L1, PD-L2 and
CD80. tDCs showed the highest level of PD-1 while lowest level of
PD-L1, as compared to other DCs (FIG. 3A). The expression PD-L2 and
CD80 have the same pattern as PD-L1 expression (FIG. 3A).
[0162] To confirm a correlation of PD-1 expression with their
functionality of T cell stimulation, a mixed lymphocytes reaction
was performed by co-culture with allogeneic total T cells isolated
from frozen PBMCs (from a different donor than in vitro generated
monocytes-derived DCs). The isolation of T cells was done by
negative selection using Pan T cell Isolation kit human (Miltenyi
Biotec, #130-096-535) following manufacturer's instructions. Total
T cells were routinely >95% pure. T cells were resuspended at
1.times.10.sup.7 cells/mL in PBS. 1 mL of Carboxyfluorescein
succinimidyl ester (CFSE Proliferation Dye, eBioscience,
#65-0850-84) at 5 nM in PBS was added per 10.sup.7 T cells
protected from light and incubated 7 minutes in a thermal bath at
37.degree. C. 10 mL of cold RPMI were added and after a wash by
centrifugation (6' at 475 g), cell number of CFSE-stained T cell
was determined using Automated cell counter Countess.TM. after a
Trypan blue coloration dilution 1 to 2. 100 .mu.L/well of medium at
37.degree. C. containing 150k T cells were seed in a 96-well plate
and incubated at 37.degree. C. in an atmosphere with 5% CO.sub.2.
100 .mu.L/well of DCs (or activated mDCs) were added to CFSE
stained T cells at the following T cell: DCs ratios; 1:30, 1:150
and 1:750. For each condition, duplicate or triplicate wells were
prepared. The co-culture was incubated at 37.degree. C. in an
atmosphere with 5% CO.sub.2. After 5 days, the plate was
centrifuged (7' at 600 g), supernatants were stored at minus
80.degree. C. were stained for T cells proliferation analysis (CFSE
dilution) by Flow Cytometry. Data presented in FIG. 3B indicate
that PD-1.sup.- mDCs are able to stimulate T cell proliferation,
whereas PD-1.sup.+ tDCs failed to activate T cells. Thus, the
expression profile of PD-1 is negatively correlated with their T
cell stimulatory capacity.
[0163] We next studied the direct effect of PD-1 inhibition on DCs.
iDCs that were treated with an anti-PD-1 mAb or anti-PD-L1 mAb led
to the upregulation of co-stimulatory molecules such as CD80, CD86,
CD83 and CD40. We then tested whether a blocking Ab of PD-L1 would
activate directly DCs to acquire enhanced T cell stimulatory
capacity. iDCs were pre-activated with or without blocking PD-L1 Ab
(10 .mu.g/mL, generated in-house at Roche, #7569), or in some
cases, with 10 .mu.g/mL of the isotype control antibody (generated
in-house at Roche; #4852) overnight for 18 hours, and extensively
washed prior to co-culture with allogenic T cells in a mixed
lymphocyte reaction for additional 5 days. Supernatant were
harvested for measurement of IFN-.gamma. by ELISA (R&D Systems,
#DY285), and the T cells were harvested for measurement of
proliferation by flow cytometry. We observed that DCs pre-incubated
with PD-L1 Ab acquired enhanced T cell stimulatory capacity (FIG.
4A), accompanied with increased IFN-.gamma. production by activated
T cells (FIG. 4B). This shows that PD-L1/PD-1 blockade-based cancer
therapy can directly target DCs to potentially promote T cell
priming and/or re-stimulation.
[0164] PD-L1 binds to both CD80 and PD-1. However the PD-L1/B7.1
interaction has a three-fold higher affinity than the CD80/CD28
interaction. To understand the complex interactions among
PD-L1/PD-1 and PD-L1/CD80, we used confocal imaging to assess their
localization on the surface of DCs. PD-1+ iDCs expressed a low
level of CD80, which doesn't co-localize with PD-1. In contrast,
PD-1- mDCs acquired higher expression of PD-L1 and CD80.
Interestingly, the CD80 now co-localized with PD-L1, suggesting
that the expression of PD-L1 in mDCs was binding to and
sequestering CD80. Thus we questioned to what extent these
molecules are involved in the formation of the DC-T-cell
immunological synapse. CD28 and PD-1 were both polarized to the
immunological synapse at the intersection of DCs and T cells. This
suggests that PD-1 and CD28 are participating within the
immunological synapse during TCR signaling. This is consistent with
the recent finding that PD-1 signaling leads to dephosphorylation
of CD28 on T cells. However, both PD-L1 and CD80 on mDCs had no or
little interaction with PD-1 and/or CD28 in the synapse. This is
again likely due to the higher binding affinity of the PD-L1/CD80
interaction versus the CD80/CD28 interaction.
[0165] The lack of strong interaction between mDC-derived CD80 and
T cell-associated CD28 prompted us to further question whether
disrupting PD-L1/CD80 interaction by a PD-L1 blocking Ab can
promote the release of CD80, making it available to CD28, leading
to co-stimulation of T cells. Indeed, pre-incubating mDCs with an
anti-PD-L1 mAb enabled strong interaction of CD80 and CD28 at the
contact of DCs and T cells. In contrast, an anti-PD-1 mAb, which
does not interfere with the binding of PD-L1 to CD80, showed no
influence on CD80 polarization. These data strongly support that an
anti-PD-L1 mAb may have two distinct effects on the ability of DCs
to activate T cells: 1.) blocking the PD-L1-mediated PD-1 signaling
on PD-1+ iDCs to promote maturation; 2) dissociating of PD-L1 from
B7.1 on mature DCs, freeing B7.1 to bind to CD28 for
co-stimulation.
Example 2: Requirement of DCs in Tumor-Bearing Mice to Respond to
PD-L1 Blockade
[0166] To confirm the physiological relevance of above in vitro
findings, we tested the anti-PD-L1 activities in in vivo animal
models. We first set up an orthotopic tumor model of C57BL/6J
female mice where PanC02-H7 (mouse pancreatic carcinoma cells
originally obtained from University of Texas M. D. Anderson Cancer
Center under a MTA) (1.times.10.sup.5 cells) were injected into the
pancreas. Seven days later, an anti-PD-L1 Ab (10 mg/kg, murine
IgG1, clone 6E11, Genentech) was administered intravenously (i.v.),
and mice were harvested at 3 days after the treatment. We analyzed
the CD11c+F4/80- DCs in the spleen and draining lymph nodes, and
found that mice received anti-PD-L1 Ab showed increased frequency
of DCs as compared to the vehicle group (FIGS. 5A-5B). Furthermore,
DCs (gated on CD11c.sup.+ cells) showed higher expression of CD86
(FIG. 5C), a marker of activation/maturation. These data suggest
that anti-PD-L1 Ab directly activates DCs in vivo.
[0167] To further study the contribution of DCs by PD-L1 blockade
in mediating anti-tumor immunity, we obtained the CD11c-DTR mice
from the Jackson laboratory (B6.FVB-Tg(Itgax-DTR/EGFP)57Lan/J, Cat.
No. 004509). CD11c-DTR mice are BALB/c mice transgenic for a high
affinity human diphtheria toxin (DT) receptor expressed under the
cd11c promoter (Jung et al., 2002, Immunity, 17: 211-20), therefore
DCs can be effectively depleted by administration of DT. Mice were
injected subcutaneously (s.c.) on study day 0 with
0.2.times.10.sup.6 of MC38 cells (colorectal cancer cell lines)
obtained from ATCC) in RPMI medium. When tumor is palpable at day
6, mice were treated with 100 ng of DT (Sigma-Aldrich) or left
untreated. At day 7, a anti-PD-L1 Ab (10 mg/kg, murine IgG1, clone
6E11, Genentech) was given to mice, followed by weekly injection
(FIG. 6A). DT treatment alone did not impact the tumor growth in
these mice (FIG. 6B). Anti-PD-L1 Ab treatment delayed tumor growth,
and had eradicated tumors in 8/10 mice, whereas in mice depleted
for DCs, anti-PD-L1 Ab efficacy was compromised significantly as
only 5/9 mice were tumor-free in the end of the study (FIG. 6B).
Our data support the concept that DCs are needed for PD-L1 Ab to
achieve its maximum anti-tumor efficacy.
Example 3: DC Gene Transcripts Predict Clinical Benefit in Patients
with Renal Cell Carcinoma Treated with Atezolizumab
[0168] We hypothesized that patients with DC abundance may respond
to PD-L1 blockade leading to beneficial effect in patients who
received the treatment. We analyzed 56 patients with renal cell
carcinoma who received Atezolizumab in a Phase I clinical trial
(NCT01375842)
(http://www.clinicaltrials.gov/ct2/show/NCT01375842?term=NCT01375842&rank-
=1). This study was sponsored by Genentech Inc., a member of the
Roche Group, which provided the study drug. The protocol and its
amendments were approved by the relevant institutional review
boards or ethics committees, and all participants provided written
informed consent. This study was conducted in accordance with the
Declaration of Helsinki and International Conference on
Harmonization Guidelines for Good Clinical Practice. In total, 56
patients were analyzed. Based on the "Best Confirmed Overall
Response by the Investigator" we observed 6 responders (CR=1; PR=5)
and 47 non-responder (PD=21; SD=26) as well as 3 patients without
known information (FIG. 7).
[0169] Tumor specimens at baseline were archived and taken for gene
expression profiling performed by RNA Sequencing (RNA-Seq). Based
on literature (Merad, ann rev immunol 2013), we selected a list of
genes consisting of XCR1, IRF8, BATF3 and FLT3, which are
associated with human dendritic cell development with
cross-presenting specialization. The log 2 RPKM expression values
of each selected gene across the whole cohort were divided at
medium expression level for the higher expression ones (+) and
lower/no expression ones (-). Using in-house R scripts the two
defined subgroups were plotted against the Kaplan-Meier survival
curves. FIGS. 8A-8B shows that every single gene expression pattern
correlates with the survival advantages. The medium survival
between patients with higher expression versus lower/no expression
were separated for at least 15.6 months or longer (FIG. 9).
[0170] As several genes linked to human dendritic cell development
were associated with the survival advantages, we investigated the
impact of multiple genes involved in human DC development and
function by defining a cumulative DC gene score (DC score)
reflecting the cumulative expression of these marker genes. Each
gene's expression is first standardized by a z-score:
z=(x-.mu.)/.sigma.,
[0171] where .mu. and .sigma. are estimated in the entire cohort or
in the selected subgroups. After the standardization step, these
standardized z-score values are averaged across genes within each
patient. The RCC cohort was corrected for the sex of patient as
well as the Stage at Initial Diagnosis. Based on such a analysis,
we observed that patients with higher DC score showed superior
survival advantage, without reaching medium survival, whereas the
lower/no expression group had a medium survival of -18 months
(HR=0.38, and p=0.03) (FIG. 10).
Example 4: DC Gene Transcripts Predict Clinical Benefit in Patients
with NSCLC Treated with Atezolizumab
[0172] Further to Example 3, we analyzed 193 patients with
non-small cell lung cancer (NSCLC) previously treated, then
received atezolizumab or docetaxel in a Phase II clinical trial
POPLAR. This study is registered with ClinicalTrials.gov, number
NCT01903993. POPLAR is a multicentre, randomised, open-label phase
II trial, done at 61 academic medical centers and community
oncology practices across 13 countries in Europe and North America.
The study was done in full accordance with the guidelines for Good
Clinical Practice and the Declaration of Helsinki. Protocol (and
modification) approval was obtained from an independent ethics
committee for each site (Fehrenbacher L, et. Al., Lancet 2016).
Among those patients with squamous or non-squamous NSCLC, 96
received Docetaxel, and 92 received atezolizumab, and the rest of 5
left untreated (Table 1).
TABLE-US-00003 TABLE 1 Patient information in POPLAR study
Treatment Squamous Non-squamous Total docetaxel 36 60 96
atezolizumab 34 58 92 not treated 1 4 5
[0173] Tumor specimens at baseline were archived and taken for gene
expression profiling performed by RNA Sequencing (RNA-Seq). Based
on literature (Merad M. et al, Ann. Rev. Immunol. 2013), we
selected a list of genes consisting of XCR1, IRF8, BATF3, and FLT3,
which are associated with human dendritic cell phenotypes and
development with cross-presenting specialization. Normalized read
counts of each selected gene across the whole cohort were divided
at medium expression level for the higher expression ones (+) and
lower/no expression ones (-). Using in-house R scripts the two
defined subgroups were plotted against the Kaplan-Meier survival
curves. In addition, Cox-regression analysis was used to calculate
the hazard ratio (HR) between the two patient groups divided based
on positive and negative gene expression.
[0174] We also investigated the impact of multiple genes involved
in human DC development and function (XCR1, BATF3, FLT3, and IRF8)
by defining a cumulative DC gene score (DC score) reflecting the
cumulative expression of these marker genes. Each gene's expression
is first standardized by a z-score:
z = x - .mu. .sigma. , ##EQU00001##
[0175] where .mu. and .sigma. are estimated in the entire cohort or
in the selected subgroups. After the standardization step, these
standardized z-score values are averaged across genes within each
patient. The cohort was corrected for the smoking status, ECOG and
sex of patient. Then the score was plotted against the Kaplan-Meier
survival curves.
[0176] Results
[0177] XCR1 gene expression pattern correlates with the survival
advantages to atezolizumab. The medium overall survival (OS)
between patients with higher expression versus lower/no expression
were separated for .about.7 months (mOS=8.6 versus 15.5 month), all
with a statistically significant hazard ratio (HR) of 0.6 (p=0.077)
by Cox-regression analysis. In contrast, there is no correlation of
XCR1 expression to survival in patients received docetaxel.
[0178] DC-related gene signature correlates with survival in
patients treated with atezolizumab as shown in the Kaplan-Meier
survival curves based on the expression of a cumulative DC gene
signature including genes: XCR1, IRF8, FLT3, and BATF3 (FIG. 11). A
higher DC signature score correlates with clinical benefit to a
PD-1 axis inhibitor atezolizumab in patients with NSCLC (HR=0.54,
p=0.04). The medium OS between patients with higher expression
versus lower/no expression were separated by 7.9 months (8.5 vs
16.4). DC-related gene signature correlates with survival in
PD-L1.sup.+ patients treated with atezolizumab as shown in the
Kaplan-Meier survival curves based on the expression of a
cumulative DC gene score in patients who are positive for PD-L1
(FIG. 12). Included in the cumulative DC gene score are XCR1, IRF8,
FLT3, and BATF3. PD-L1 expression was assessed prospectively on
tumor cells and tumor-infiltrating immune cells with the VENTANA
SP142 PD-L1 immunohistochemistry assay (Ventana Medical Systems,
Tucson, Ariz., USA) (Fehrenbacher L, et. Al., Lancet 2016).
Expressing of PD-L1 was scored as a percentage of total tumor cells
and tumor-infiltrating immune cells expressing PD-L1 as a
percentage of tumor area (tumor cells scored as percentage of
PD-L1-expressing tumor cells: TC3.gtoreq.50%, TC2.gtoreq.5% and
.ltoreq.50%, TC1.gtoreq.1% and .ltoreq.5%, and TC0.ltoreq.1%;
tumor-infiltrating immune cells scored as percentage of tumor area:
IC3.gtoreq.50%, IC2.gtoreq.5% and .ltoreq.50%, IC1.gtoreq.1% and
.ltoreq.5%, and IC0.ltoreq.1%). We considered PD-L1+ patients
grouped as TC3, TC2, IC3, and IC2. A strong correlation of DC
signature score and clinical benefit to a PD-1 axis inhibitor
atezolizumab was observed in PD-L1.sup.+ patients (HR=0.25,
p=0.03). The medium OS in patients with higher DC gene score was
not reached, whereas in patients with lower/no expression has
median OS of 8.4 months. In contrast, there is no correlation of DC
genes expression to survival in PD-L1.sup.+ patients received
docetaxel.
Sequence CWU 1
1
10110PRTArtificial SequenceHVR-H1 1Gly Phe Thr Phe Ser Asp Ser Trp
Ile His1 5 10218PRTArtificial SequenceHVR-H2 2Ala Trp Ile Ser Pro
Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val1 5 10 15Lys
Gly39PRTArtificial SequenceHVR-H3 3Arg His Trp Pro Gly Gly Phe Asp
Tyr1 5411PRTArtificial SequenceHVR-L1 4Arg Ala Ser Gln Asp Val Ser
Thr Ala Val Ala1 5 1057PRTArtificial SequenceHVR-L2 5Ser Ala Ser
Phe Leu Tyr Ser1 569PRTArtificial SequenceHVR-L3 6Gln Gln Tyr Leu
Tyr His Pro Ala Thr1 57118PRTArtificial SequenceVH 7Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30Trp Ile
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala
Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65
70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln
Gly Thr 100 105 110Leu Val Thr Val Ser Ser 1158108PRTArtificial
SequenceVL 8Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val
Ser Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Tyr Leu Tyr His Pro Ala 85 90 95Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg 100 1059447PRTArtificial SequenceHC 9Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25
30Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr
Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr
Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170
175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp Lys Thr 210 215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser225 230 235 240Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val 290 295
300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr305 310 315 320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr 325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu 340 345 350Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys 355 360 365Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390 395 400Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410
415Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly 435 440 44510214PRTArtificial SequenceLC 10Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30Val Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser
Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 210
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References